Nanopore Direct RNA Sequencing Reveals Virus-Induced Changes in the Transcriptional Landscape in Human Bronchial Epithelial Cells.

Direct RNA nanopore sequencing reveals changes in gene expression, polyadenylation, splicing, m6A methylation, and pseudouridylation in response to influenza virus exposure in primary human bronchial epithelial cells. This study focuses on the epitranscriptomic profile of genes in the host immune response. In addition to polyadenylated noncoding RNA, we purified and sequenced nonpolyadenylated noncoding RNA and observed changes in expression, N6-methyl-adenosine (m6A), and pseudouridylation (Ψ) in these novel RNA. Two recently discovered lincRNA with roles in immune response, Chaserr and LEADR , became highly methylated in response to influenza exposure. Several H/ACA type snoRNAs that guide pseudouridylation are decreased in expression in response to influenza, and there is a corresponding decrease in the pseudouridylation of two novel lncRNA. Thus, novel epitranscriptomic changes revealed by direct RNA sequencing with nanopore technology provides unique insights into the host epitranscriptomic changes in epithelial gene networks that respond to influenza virus infection.

Influenza virus infection exacerbates gene expression related to neurocognitive dysfunction in brains of old mice.

BACKGROUND: Age > 65 years is a key risk factor for poor outcomes after human influenza infection. Specifically, in addition to respiratory disease, non-neurotropic influenza A virus (IAV) causes neuro-cognitive complications, e.g. new onset depression and increases the risk of dementia after hospitalization. This study aimed to identify potential mechanisms of these effects by determining differences between young and old mice in brain gene expression in a mouse model of non-neurotropic IAV infection. METHODS: Young (12 weeks) and old (70 weeks) C57Bl/6J mice were inoculated intranasally with 200 PFU H1N1 A/PR/34/8 (PR8) or sterile PBS (mock). Gene expression in lung and brain was measured by qRT-PCR and normalized to ß-actin. Findings were confirmed using the nCounter Mouse Neuroinflammation Array (NanoString) and analyzed with nSolver 4.0 and Ingenuity Pathway Analysis (IPA, Qiagen). RESULTS: IAV PR8 did not invade the central nervous system. Young and old mice differed significantly in brain gene expression at baseline and during non-neurotropic IAV infection. Expression of brain Ifnl, Irf7, and Tnf mRNAs was upregulated over baseline control at 3 days post-infection (p.i.) only in young mice, but old mice expressed more Ifnl than young mice 7 days p.i. Gene arrays showed down-regulation of the Epigenetic Regulation, Insulin Signaling, and Neurons and Neurotransmission pathways in old mice 3 days p.i. while young mice demonstrated no change or induction of these pathways at the same time point. IPA revealed marked baseline differences between old and young mice. Gene expression related to Cognitive Impairment, Memory Deficits and Learning worsened in old mice relative to young mice during IAV infection. Aged mice demonstrate more severe changes in gene expression related to memory loss and cognitive dysfunction by IPA. CONCLUSIONS: These data suggest the genes and pathways related to learning and cognitive performance that were worse at baseline in old mice were further worsened by IAV infection, similar to old patients. Early events in the brain triggered by IAV infection portend downstream neurocognitive pathology in old adults.

Bacillus anthracis spores are internalized in human lung epithelial cells by Rab GTPase-supported macropinocytosis.

Inhalation anthrax, the deadliest form of the disease, requires inhaled B. anthracis spores to escape from the alveolar space and travel to the mediastinal lymph nodes, from where the vegetative form of the pathogen disseminates, resulting in a rapidly fatal outcome. The role of epithelia in alveolar escape is unclear, but previous work suggests these epithelial cells are involved in this process. Using confocal microscopy, we found that B. anthracis spores are internalized more rapidly by A549 type II alveolar epithelial cells compared to hAELVi type I alveolar epithelial cells. Internalization of spores by alveolar epithelial cells requires cytoskeletal rearrangement evidenced by significant inhibition by cytochalasin D, an actin inhibitor. Chemical inhibitors of macropinocytosis significantly downregulated B. anthracis spore internalization in human alveolar cells, while inhibitors of other endocytosis pathways had minimal effects. Additional studies using a macropinosome marker and electron microscopy confirmed the role of macropinocytosis in spore uptake. By colocalization of B. anthracis spores with four endocytic Rab proteins, we demonstrated that Rab31 played a role in B. anthracis spore macropinocytosis. Finally, we confirmed that Rab31 is involved in B. anthracis spore internalization by enhanced spore uptake in Rab31-overexpressing A549 cells. This is the first report that shows B. anthracis spore internalization by macropinocytosis in human epithelial cells. Several Rab GTPases are involved in the process.

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

Rationale: A 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. Methods: Mice 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. Results: SLR10 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. Conclusions: Our 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 interaction of human phagocytic APC subsets with Cryptococcus neoformans induces genes associated with metabolism and antigen presentation.

Cryptococcal meningitis is the most common cause of meningitis among HIV/AIDS patients in sub-Saharan Africa, and worldwide causes over 223,000 cases leading to more than 181,000 annual deaths. Usually, the fungus gets inhaled into the lungs where the initial interactions occur with pulmonary phagocytes such as dendritic cells and macrophages. Following phagocytosis, the pathogen can be killed or can replicate intracellularly. Previous studies in mice showed that different subsets of these innate immune cells can either be antifungal or permissive for intracellular fungal growth. Our studies tested phagocytic antigen-presenting cell (APC) subsets from the human lung against C. neoformans. Human bronchoalveolar lavage was processed for phagocytic APCs and incubated with C. neoformans for two hours to analyze the initial interactions and fate of the fungus, living or killed. Results showed all subsets (3 macrophage and 3 dendritic cell subsets) interacted with the fungus, and both living and killed morphologies were discernable within the subsets using imaging flow cytometry. Single cell RNA-seq identified several different clusters of cells which more closely related to interactions with C. neoformans and its protective capacity against the pathogen rather than discrete cellular subsets. Differential gene expression analyses identified several changes in the innate immune cell's transcriptome as it kills the fungus including increases of TNF-α (TNF) and the switch to using fatty acid metabolism by upregulation of the gene FABP4. Also, increases of TNF-α correlated to cryptococcal interactions and uptake. Together, these analyses implicated signaling networks that regulate expression of many different genes - both metabolic and immune - as certain clusters of cells mount a protective response and kill the pathogen. Future studies will examine these genes and networks to understand the exact mechanism(s) these phagocytic APC subsets use to kill C. neoformans in order to develop immunotherapeutic strategies to combat this deadly disease.

Early IFN-ß administration protects cigarette smoke exposed mice against lethal influenza virus infection without increasing lung inflammation.

During influenza A virus (IAV) infection, it is unclear whether type I interferons (IFNs) have defensive antiviral effects or contribute to immunopathology in smokers. We treated nonsmoking (NS) and cigarette smoke (CS)-exposed mice intranasally with early (prophylactic) or late (therapeutic) IFN-ß. We compared the mortality and innate immune responses of the treated mice following challenge with IAV. In NS mice, both early and late IFN-ß administration decreased the survival rate in mice infected with IAV, with late IFN-ß administration having the greatest effect on survival. In contrast, in CS-exposed mice, early IFN-ß administration significantly increased survival during IAV infection while late IFN-ß administration did not alter mortality. With regards to inflammation, in NS mice, IFN-ß administration, especially late administration, significantly increased IAV-induced inflammation and lung injury. Early IFN-ß administration to CS-exposed mice did not increase IAV-induced inflammation and lung injury as occurred in NS mice. Our results demonstrate, although IFN-ß administration worsens the susceptibility of NS mice to influenza infection with increased immunopathology, early IFN-ß administration to CS-exposed mice, which have suppression of the intrinsic IFN response, improved outcomes during influenza infection.

Long-term cigarette smoke exposure dysregulates pulmonary T cell response and IFN-γ protection to influenza virus in mouse.

BACKGROUND: Influenza is a highly contagious, acute, febrile respiratory infection caused by a negative-sense, single-stranded RNA virus, which belongs in the Orthomyxoviridae family. Cigarette smoke (CS) exposure worsens influenza infection in terms of frequency and severity in both human and animal models. METHODS: C57BL/6 mice with or without CS exposure for 6 weeks were inoculated intranasally with a single, non-lethal dose of the influenza A virus (IAV) A/Puerto Rico/8/1934 (PR8) strain. At 7 and 10 days after infection, lung and mediastinal lymph nodes (MLN) cells were collected to determine the numbers of total CD4 + and CD8 + T cells, and IAV-specific CD4 + and CD8 + T cells, using flow cytometry. Bronchoalveolar lavage fluid (BALF) was also collected to determine IFN-γ levels and total protein concentration. RESULTS: Although long-term CS exposure suppressed early pulmonary IAV-antigen specific CD8 + and CD4 + T cell numbers and IFN-γ production in response to IAV infection on day 7 post-infection, CS enhanced numbers of these cells and IFN-γ production on day 10. The changes of total protein concentration in BALF are consistent with the changes in the IFN-γ amounts between day 7 and 10, which suggested that excessive IFN-γ impaired barrier function and caused lung injury at the later stage of infection. CONCLUSIONS: Our results demonstrated that prior CS exposure caused a biphasic T cell and IFN-γ response to subsequent infection with influenza in the lung. Specifically, the number of IAV antigen-specific T cells on day 10 was greatly increased by CS exposure even though CS decreased the number of the same group of cells on day 7. The result suggested that CS affected the kinetics of the T cell response to IAV, which was suppressed at an early stage and exaggerated at a later stage. This study is the first to describe the different effect of long-term CS on T cell responses to IAV at early and late stages of infection in vivo.

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 Signaling via MAVS Is Dispensable for Survival in Lethal Influenza Infection In Vivo.

Retinoic acid-inducible gene I (RIG-I) is an important regulator of virus-induced antiviral interferons (IFNs) and proinflammatory cytokines. It requires interaction with an adaptor molecule, mitochondrial antiviral-signaling protein (MAVS), to activate downstream signaling pathways. To elucidate the mechanism(s) by which RIG-I-dependent recognition of IAV infection in vivo triggers innate immune responses, we infected mutant mice lacking RIG-I or MAVS with influenza A virus (IAV) and measured their innate immune responses. As has previously been demonstrated with isolated deletion of the virus recognition receptors TLR3, TLR7, and NOD2, RIG-I or MAVS knockout (KO) did not result in higher mortality and did not reduce IAV-induced cytokine responses in mice. Infected RIG-I KO animals displayed similar lung inflammation profiles as did WT mice, in terms of the protein concentration, total cell count, and inflammatory cell composition in the bronchoalveolar lavage fluid. RNA-Seq results demonstrated that all types of mice exhibited equivalent antiviral and inflammatory gene responses following IAV infection. Together, the results indicated that although RIG-I is important in innate cytokine responses in vitro, individual deletion of the genes encoding RIG-I or MAVS did not change survival or innate responses in vivo after IAV infection in mice.

Gene expression profiling of primary human type I alveolar epithelial cells exposed to Bacillus anthracis spores reveals induction of neutrophil and monocyte chemokines.

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 h post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 h and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1ß (4 h), CXCL8/IL-8 (4 and 24 h) and CXCL5/ENA-78 (24 h). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROß and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.

Transcriptional Classification and Functional Characterization of Human Airway Macrophage and Dendritic Cell Subsets.

The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cells that work together to maintain steady-state respiration. Owing to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systematically identify these subsets in human airways by developing a schema of isolating large numbers of cells by whole-lung bronchoalveolar lavage. Six subsets of phagocytic APC (HLA-DR+) were consistently observed. Aside from alveolar macrophages, subsets of Langerin+, BDCA1-CD14+, BDCA1+CD14+, BDCA1+CD14-, and BDCA1-CD14- cells were identified. These subsets varied in their ability to internalize Escherichia coli, Staphylococcus aureus, and Bacillus anthracis particles. All subsets were more efficient at internalizing S. aureus and B. anthracis compared with E. coli Alveolar macrophages and CD14+ cells were overall more efficient at particle internalization compared with the four other populations. Subsets were further separated into two groups based on their inherent capacities to upregulate surface CD83, CD86, and CCR7 expression levels. Whole-genome transcriptional profiling revealed a clade of "true dendritic cells" consisting of Langerin+, BDCA1+CD14+, and BDCA1+CD14- cells. The dendritic cell clade was distinct from a macrophage/monocyte clade, as supported by higher mRNA expression levels of several dendritic cell-associated genes, including CD1, FLT3, CX3CR1, and CCR6 Each clade, and each member of both clades, was discerned by specific upregulated genes, which can serve as markers for future studies in healthy and diseased states.

Human primary airway epithelial cells isolated from active smokers have epigenetically impaired antiviral responses.

BACKGROUND: Cigarette smoking (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and most COPD exacerbations are caused by respiratory infections including influenza. Influenza infections are more severe in smokers. The mechanism of the increased risk and severity of infections in smokers is likely multifactorial, but certainly includes changes in immunologic host defenses. METHODS: We investigated retinoic acid-inducible protein I (RIG-I) and interferon (IFN) induction by influenza A virus (IAV) in human bronchial epithelial cells (HBEC) isolated from smokers or nonsmokers. Subcultured HBEC cells were infected with A/Puerto Rico/8/1934 (PR8) IAV at an MOI of 1. After 24 h of infection, cells and supernatants were collected for qRT-PCR, immunoblot or ELISA to determine RIG-I, Toll-like receptor3 (TLR3) and IFN expression levels. RESULTS: IAV exposure induced a vigorous IFN-ß, IFN-λ 1 and IFN-λ 2/3 antiviral response in HBEC from nonsmokers and significant induction of RIG-I and TLR3. In cells from smokers, viral RIG-I and TLR3 mRNA induction was reduced 87 and 79 % compared to the response from nonsmokers. CS exposure history was associated with inhibition of viral induction of the IFN-ß, IFN-λ1 and IFN-λ 2/3 mRNA response by 85, 96 and 95 %, respectively, from that seen in HBEC from nonsmokers. The demethylating agent 5-Aza-2-deoxycytidine reversed the immunosuppressive effects of CS exposure in HBEC since viral induction of all three IFNs was restored. IFN-ß induction of RIG-I and TLR3 was also suppressed in the cells from smokers. CONCLUSION: Our results suggest that active smoking reduces expression of antiviral cytokines in primary HBEC cells. This effect likely occurs via downregulation of RIG-I and TLR3 due to smoke-induced epigenetic modifications. Reduction in lung epithelial cell RIG-I and TLR3 responses may be a major mechanism contributing to the increased risk and severity of viral respiratory infections in smokers and to viral-mediated acute exacerbations of COPD.

Bacillus anthracis spore movement does not require a carrier cell and is not affected by lethal toxin in human lung models.

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores escape from the alveolus to regional lymph nodes, germinate and enter the circulatory system to cause disease. The roles of carrier cells and the effects of B. anthracis toxins in this process are unclear. We used a human lung organ culture model to measure spore uptake by antigen presenting cells (APC) and alveolar epithelial cells (AEC), spore partitioning between these cells, and the effects of B. anthracis lethal toxin and protective antigen. We repeated the study in a human A549 alveolar epithelial cell model. Most spores remained unassociated with cells, but the majority of cell-associated spores were in AEC, not in APC. Spore movement was not dependent on internalization, although the location of internalized spores changed in both cell types. Spores also internalized in a non-uniform pattern. Toxins affected neither transit of the spores nor the partitioning of spores into AEC and APC. Our results support a model of spore escape from the alveolus that involves spore clustering with transient passage through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.

RIG-I and TLR3 are both required for maximum interferon induction by influenza virus in human lung alveolar epithelial cells.

Pattern recognition receptors, such as retinoic acid-inducible protein I (RIG-I), Toll-like receptors 3 and 7 (TLR3 and 7), and nucleotide-binding oligomerization domain containing protein 2 (NOD2), play important roles in the recognition of influenza A virus (IAV), but their role in interferon (IFN) induction is still unclear, particularly in human lung. We investigated IFN induction by IAV in the A549 cell line as well as in primary human alveolar epithelial cells (AEC). TLR3/7, NOD2, RIG-I, and IFN expression levels were measured by qRT-PCR and ELISA in cells infected with IAV PR8. We found that TLR7 and NOD2 were not involved in IFN induction by IAV in these cells. Neither RIG-I nor TLR3 siRNA alone completely blocked IFN induction. However, double knockdown of RIG-I and TLR3 completely inhibited IFN induction by influenza. Thus, signaling through both RIG-I and TLR3 is important for IFN induction by IAV in human lung AEC.

Cigarette smoke attenuates the RIG-I-initiated innate antiviral response to influenza infection in two murine models.

Cigarette smoke (CS) exposure increases the frequency and severity of respiratory tract infections. Despite this association, the mechanisms underlying the increased susceptibility to respiratory virus infection are poorly understood. Retinoic acid-inducible gene I (RIG-I) is an important regulator of influenza virus-induced expression of antiviral cytokines, mainly interferons (IFNs), which are necessary to clear viral infections. In this study, we compared the innate cytokine responses of two mouse CS exposure models following a challenge with influenza A virus (IAV): 1) exposure of the mice to cigarette smoke extract (CSE) intratracheally and 2) exposure of the mice to CS in a whole body exposure chamber. Both intratracheal CSE treatment and whole body CS exposure caused antiviral immunosuppression in these mice, and both CS exposure methods inhibited RIG-I induction. CS attenuated influenza-induced antiviral IFNs and IP-10 expression in vivo. However, we did not find that CS inhibited induction of the proinflammatory cytokines IL-6 and TNF-α, whose expression was induced by IAV. Interestingly, IAV infection also increased Toll-like receptor 3 (TLR3) expression in mouse lung, but CS exposure did not impact TLR3 induction in these mice. Together, the results support our previous finding in a human lung organ culture model that the suppression of RIG-I induction and antiviral cytokine responses by CS are likely important in the enhanced susceptibility of smokers to influenza infection in the lung.

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-ß (IFN-ß) mRNA induction by the two viruses. RIG-I and IFN-ß 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.

Cigarette smoke extract suppresses the RIG-I-initiated innate immune response to influenza virus in the human lung.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and predisposes subjects to severe respiratory tract infections. Epidemiological studies have shown that cigarette smokers are seven times more likely to contract influenza infection than nonsmokers. The mechanisms underlying this increased susceptibility are poorly characterized. Retinoic acid-inducible gene (RIG)-I is believed to play an important role in the recognition of, and response to, influenza virus and other RNA viruses. Our study focused on how cigarette smoke extract (CSE) alters the influenza-induced proinflammatory response and suppresses host antiviral activity in the human lung using a unique lung organ culture model. We first determined that treatment with 2-20% CSE did not induce cytotoxicity as assessed by LDH release. However, CSE treatment inhibited influenza-induced IFN-inducible protein 10 protein and mRNA expression. Induction of the major antiviral cytokine IFN-ß mRNA was also decreased by CSE. CSE also blunted viral-mediated RIG-I mRNA and protein expression. Inhibition of viral-mediated RIG-I induction by CSE was prevented by the antioxidants N-acetyl-cysteine and glutathione. These findings show that CSE suppresses antiviral and innate immune responses in influenza virus-infected human lungs through oxidative inhibition of viral-mediated induction of the pattern recognition receptor RIG-I. This immunosuppressive effect of CSE may play a role in the enhanced susceptibility of smokers to serious influenza infection in the lung.

Human lung innate immune cytokine response to adenovirus type 7.

Adenovirus (Ad) type 7 can cause severe infection, including pneumonia, in military recruits and children. The initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. Subsequently, monocytes become evident and, finally, there is a predominantly lymphocytic infiltrate. We have established that Ad7 infection of epithelial cells stimulates release of the neutrophil chemotaxin interleukin (IL)-8, and have extended these studies to a human lung tissue model. Here, we studied cytokine responses to Ad7 in human alveolar macrophages (HAM) and our human lung tissue model. Both ELISA and RNase-protection assay (RPA) data demonstrated that, upon Ad7 infection, IP-10 and MIP-1alpha/beta are released from HAM. IP-10 and MIP-1alpha/beta protein levels were induced 2- and 3-fold, respectively, in HAM 24 h after Ad7 infection. We then investigated induction of specific cytokines in human lung tissue by RPA and ELISA. The results showed that IL-8 and IL-6 were induced 8 h after infection and, by 24 h, levels of IL-8, IL-6, MIP-1alpha/beta and MCP-1 were all increased. IP-10, a monocyte and lymphocyte chemokine, was also induced 30-fold, but only 24 h after infection. Immunohistochemistry staining confirmed that IL-8 was only released from the epithelial cells of lung slices and not from macrophages. IP-10 was secreted from both macrophages and epithelial cells. Moreover, full induction of IP-10 is likely to require participation and cooperation of both epithelial cells and macrophages in intact lung. Understanding the cytokine and chemokine induction during Ad7 infection may lead to novel ways to modulate the response to this pathogen.

Innate immune response to H3N2 and H1N1 influenza virus infection in a human lung organ culture model.

We studied cytokine responses to influenza virus PR8 (H1N1) and Oklahoma/309/06 (OK/06, H3N2) in a novel human lung tissue model. Exposure of the model to influenza virus rapidly activated the mitogen-activated protein kinase signaling (MAPK) pathways ERK, p38 and JNK. In addition, RNase protection assay demonstrated the induction of several cytokine and chemokine mRNAs by virus. This finding was reflected at the translational level as IL-6, MCP-1, MIP-1 alpha/beta, IL-8 and IP-10 proteins were induced as determined by ELISA. Immunohistochemistry for IP-10 and MIP-1 alpha revealed that alveolar epithelial cells and macrophages were the source of these two cytokines. Taken together, both PR8 and OK/06 cause similar induction of cytokines in human lung, although OK/06 is less effective at inducing the chemokines MCP-1 and IL-8. This human organ culture model should thus provide a relevant platform to study the biological responses of human lung to influenza virus infection.

Resistance of human alveolar macrophages to Bacillus anthracis lethal toxin.

The etiologic agent of inhalational anthrax, Bacillus anthracis, produces virulence toxins that are important in the disease pathogenesis. Current studies suggest that mouse and human macrophages are susceptible to immunosuppressive effects of one of the virulence toxins, lethal toxin (LT). Thus a paradigm has emerged that holds that the alveolar macrophage (AM) does not play a significant role in the innate immune response to B. anthracis or defend against the pathogen as it is disabled by LT. This is inconsistent with animal models and autopsy studies that show minimal disease at the alveolar surface. We examined whether AM are immunosuppressed by LT. We found that human AM were relatively resistant to LT-mediated innate immune cytokine suppression, MEK cleavage, and induction of apoptosis as compared with mouse RAW 264.7 macrophages. Mouse AM and murine bone marrow-derived macrophages were also relatively resistant to LT-mediated apoptosis despite intermediate sensitivity to MEK cleavage. The binding component of LT, protective Ag, does not attach to human AM, although it did bind to mouse AM, murine bone marrow-derived macrophages, and RAW 264.7 macrophages. Human AM do not produce significant amounts of the protective Ag receptor anthrax toxin receptor 1 (TEM8/ANTXR1) and anthrax toxin receptor 2 (CMG2/ANTXR2). Thus, mature and differentiated AM are relatively resistant to the effects of LT as compared with mouse RAW 264.7 macrophages. AM resistance to LT may enhance clearance of the pathogen from the alveolar surface and explain why this surface is relatively free of B. anthracis in animal models and autopsy studies.