Gene expression profiling of human alveolar macrophages infected by B. anthracis spores demonstrates TNF-α and NF-κb are key components of the innate immune response to the pathogen

<p>Abstract</p> <p>Background</p> <p><it>Bacillus anthracis</it>, the etiologic agent of anthrax, has recently been used as an agent of bioterrorism. The innate immune system initially appears to contain the pathogen at the site of entry. Because the human alveolar macrophage (HAM) plays a key role in lung innate immune responses, studying the HAM response to <it>B. anthracis </it>is important in understanding the pathogenesis of the pulmonary form of this disease.</p> <p>Methods</p> <p>In this paper, the transcriptional profile of <it>B. anthracis </it>spore-treated HAM was compared with that of mock-infected cells, and differentially expressed genes were identified by Affymetrix microarray analysis. A portion of the results were verified by Luminex protein analysis.</p> <p>Results</p> <p>The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The differentially expressed genes were subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, the Promoter Analysis and Interaction Network Toolset (PAINT) and Oncomine analysis. Among the upregulated genes, we identified a group of chemokine ligand, apoptosis, and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-α, NF-κB and their ligands/receptors. In addition to TNF-α, a broad range of cytokines was induced, and this was confirmed at the level of translation by Luminex multiplex protein analysis. PAINT analysis revealed that many of the genes affected by spores contain the binding site for c-Rel, a member of the NF-κB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. However, many of the genes are poorly annotated, indicating that they represent novel functions. Four of the genes most highly regulated by spores have only previously been associated with head and neck and lung carcinomas.</p> <p>Conclusion</p> <p>The results demonstrate not only that TNF-α and NF-κb are key components of the innate immune response to the pathogen, but also that a large part of the mechanisms by which the alveolar macrophage responds to <it>B. anthracis </it>are still unknown as many of the genes involved are poorly annotated.</p

RIG-I agonist SLR10 promotes macrophage M1 polarization during influenza virus infection

RationaleA family of short synthetic, triphosphorylated stem-loop RNAs (SLRs) have been designed to activate the retinoic-acid-inducible gene I (RIG-I) pathway and induce a potent interferon (IFN) response, which may have therapeutic potential. We investigated immune response modulation by SLR10. We addressed whether RIG-I pathway activation with SLR10 leads to protection of nonsmoking (NS) and cigarette smoke (CS)-exposed mice after influenza A virus (IAV) infection.MethodsMice were given 25 µg of SLR10 1 day before IAV infection. We compared the survival rates and host immune responses of NS and CS-exposed mice following challenge with IAV.ResultsSLR10 significantly decreased weight loss and increased survival rates in both NS and CS-exposed mice during IAV infection. SLR10 administration repaired the impaired proinflammatory response in CS-exposed mice without causing more lung injury in NS mice as assessed by physiologic measurements. Although histopathologic study revealed that SLR10 administration was likely to result in higher pathological scores than untreated groups in both NS and CS mice, this change was not enough to increase lung injury evaluated by lung-to-body weight ratio. Both qRT-PCR on lung tissues and multiplex immunoassay on bronchoalveolar lavage fluids (BALFs) showed that most IFNs and proinflammatory cytokines were expressed at lower levels in SLR10-treated NS mice than control-treaded NS mice at day 5 post infection (p.i.). Remarkably, proinflammatory cytokines IL-6, IL-12, and GM-CSF were increased in CS-exposed mice by SLR10 at day 5 p.i. Significantly, SLR10 elevated the ratio of the two chemokines (CXCL9 and CCL17) in BALFs, suggesting macrophages were polarized to classically activated (M1) status. In vitro testing also found that SLR10 not only stimulated human alveolar macrophage polarization to an M1 phenotype, but also reversed cigarette smoke extract (CSE)-induced M2 to M1 polarization.ConclusionsOur data show that SLR10 administration in mice is protective for both NS and CS-exposed IAV-infected mice. Mechanistically, SLR10 treatment promoted M1 macrophage polarization in the lung during influenza infection. The protective effects by SLR10 may be a promising intervention for therapy for infections with viruses, particularly those with CS-enhanced susceptibility to adverse outcomes

Protective essential oil attenuates influenza virus infection: An in vitro study in MDCK cells

<p>Abstract</p> <p>Background</p> <p>Influenza is a significant cause of morbidity and mortality. The recent pandemic of a novel H1N1 influenza virus has stressed the importance of the search for effective treatments for this disease. Essential oils from aromatic plants have been used for a wide variety of applications, such as personal hygiene, therapeutic massage and even medical practice. In this paper, we investigate the potential role of an essential oil in antiviral activity.</p> <p>Methods</p> <p>We studied a commercial essential oil blend, On Guard™, and evaluated its ability in modulating influenza virus, A/PR8/34 (PR8), infection in Madin-Darby canine kidney (MDCK) cells. Influenza virus was first incubated with the essential oil and infectivity in MDCK cells was quantified by fluorescent focus assay (FFA). In order to determine the mechanism of effects of essential oil in viral infection inhibition, we measured hemagglutination (HA) activity, binding and internalization of untreated and oil-treated virus in MDCK cells by flow cytometry and immunofluorescence microscopy. In addition, the effect of oil treatment on viral transcription and translation were assayed by relative end-point RT-PCR and western blot analysis.</p> <p>Results</p> <p>Influenza virus infectivity was suppressed by essential oil treatment in a dose-dependent manner; the number of nascent viral particles released from MDCK cells was reduced by 90% and by 40% when virus was treated with 1:4,000 and 1:6,000 dilutions of the oil, respectively. Oil treatment of the virus also decreased direct infection of the cells as the number of infected MDCK cells decreased by 90% and 45% when virus was treated with 1:2,000 and 1:3,000 dilutions of the oil, respectively. This was not due to a decrease in HA activity, as HA was preserved despite oil treatment. In addition, oil treatment did not affect virus binding or internalization in MDCK cells. These effects did not appear to be due to cytotoxicity of the oil as MDCK cell viability was only seen with concentrations of oil that were 2 to 6 times greater than the doses that inhibited viral infectivity. RT-PCR and western blotting demonstrated that oil treatment of the virus inhibited viral NP and NS1 protein, but not mRNA expression.</p> <p>Conclusions</p> <p>An essential oil blend significantly attenuates influenza virus PR8 infectivity <it>in vitro </it>without affecting viral binding or cellular internalization in MDCK cells. Oil treated virus continued to express viral mRNAs but had minimal expression of viral proteins, suggesting that the antiviral effect may be due to inhibition of viral protein translation.</p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Influenza A(H1N1)pdm09 virus suppresses RIG-I initiated innate antiviral responses in the human lung.

Influenza infection is a major cause of morbidity and mortality. Retinoic acid-inducible gene I (RIG-I) is believed to play an important role in the recognition of, and response to, influenza virus and other RNA viruses. Our study focuses on the hypothesis that pandemic H1N1/09 influenza virus alters the influenza-induced proinflammatory response and suppresses host antiviral activity. We first compared the innate response to a clinical isolate of influenza A(H1N1)pdm09 virus, OK/09, a clinical isolate of seasonal H3N2 virus, OK/06, and to a laboratory adapted seasonal H1N1 virus, PR8, using a unique human lung organ culture model. Exposure of human lung tissue to either pandemic or seasonal influenza virus resulted in infection and replication in alveolar epithelial cells. Pandemic virus induces a diminished RIG-I mRNA and antiviral cytokine response than seasonal virus in human lung. The suppression of antiviral response and RIG-I mRNA expression was confirmed at the protein level by ELISA and western blot. We performed a time course of RIG-I and interferon-β (IFN-β) mRNA induction by the two viruses. RIG-I and IFN-β induction by OK/09 was of lower amplitude and shorter duration than that caused by PR8. In contrast, the pandemic virus OK/09 caused similar induction of proinflammatory cytokines, IL-8 and IL-6, at both the transcriptional and translational level as PR8 in human lung. Differential antiviral responses did not appear to be due to a difference in cellular infectivity as immunohistochemistry showed that both viruses infected alveolar macrophages and epithelial cells. These findings show that influenza A(H1N1)pdm09 virus suppresses anti-viral immune responses in infected human lung through inhibition of viral-mediated induction of the pattern recognition receptor, RIG-I, though proinflammatory cytokine induction was unaltered. This immunosuppression of the host antiviral response by pandemic virus may have contributed to the more serious lung infections that occurred in the H1N1 pandemic of 2009

Anthrax Edema and Lethal Toxins Differentially Target Human Lung and Blood Phagocytes

Bacillus anthracis, the causative agent of inhalation anthrax, is a serious concern as a bioterrorism weapon. The vegetative form produces two exotoxins: Lethal toxin (LT) and edema toxin (ET). We recently characterized and compared six human airway and alveolar-resident phagocyte (AARP) subsets at the transcriptional and functional levels. In this study, we examined the effects of LT and ET on these subsets and human leukocytes. AARPs and leukocytes do not express high levels of the toxin receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). Less than 20% expressed surface TEM8, while less than 15% expressed CMG2. All cell types bound or internalized protective antigen, the common component of the two toxins, in a dose-dependent manner. Most protective antigen was likely internalized via macropinocytosis. Cells were not sensitive to LT-induced apoptosis or necrosis at concentrations up to 1000 ng/mL. However, toxin exposure inhibited B. anthracis spore internalization. This inhibition was driven primarily by ET in AARPs and LT in leukocytes. These results support a model of inhalation anthrax in which spores germinate and produce toxins. ET inhibits pathogen phagocytosis by AARPs, allowing alveolar escape. In late-stage disease, LT inhibits phagocytosis by leukocytes, allowing bacterial replication in the bloodstream

RIG-I and IP-10 induction in epithelial cells in the human lung.

<p>Lung slices were exposed to 6×10⁶ PFU/ml of influenza virus PR8 or OK/09 or virus diluents for 24 h in the presence of BFA to enhance the detection of cytokines. Slices were then processed for immunohistochemistry for the detection of IP-10 using goat polyclonal antibodies, RIG-I using rabbit polyclonal antibody, and epithelial cells using anti-cytokeratin monoclonal antibody. <i>Top</i>: OK/09. <i>Bottom</i>: PR8. <i>A–D</i>: fluorescent images that demonstrate nuclei (<i>A</i>; blue), RIG-I (<i>B</i>; red), IP-10 (<i>C</i>; green), and epithelial cells (<i>D</i>; cyan). <i>E</i>: bright-field images. <i>F</i>: overlays of the fluorescent images that demonstrate that influenza induces RIG-I in epithelial cells (arrows). Bars = 100 µm.</p

Pandemic OK/09 induces lower RIG-I protein responses than PR8 in human lung.

<p>Human lung slices were exposed to 6×10⁶ PFU/ml of PR8 or OK/09 for 8 h and 24 h. Western blot analysis was used to determine RIG-I protein expression in lung slices. Membranes were probed with anti-RIG-I or anti-GAPDH antibodies. Protein expression of RIG-I was normalized relative to GAPDH. Data are representative of 3 separate experiments.</p