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2.
Nature ; 587(7834): 466-471, 2020 11.
Article in English | MEDLINE | ID: mdl-33116313

ABSTRACT

Severe respiratory infections can result in acute respiratory distress syndrome (ARDS)1. There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS1,2. Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes-in particular the ECM protease ADAMTS4-and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections.


Subject(s)
ADAMTS4 Protein/metabolism , Fibroblasts/enzymology , Fibroblasts/pathology , Influenza A virus/pathogenicity , Lung/pathology , Lung/physiopathology , ADAMTS4 Protein/antagonists & inhibitors , Animals , Birds/virology , Extracellular Matrix/enzymology , Gene Expression Profiling , Humans , Influenza in Birds/virology , Influenza, Human/pathology , Influenza, Human/therapy , Influenza, Human/virology , Interferons/immunology , Interferons/metabolism , Leukocyte Common Antigens/metabolism , Lung/enzymology , Lung/virology , Mice , Respiratory Distress Syndrome/enzymology , Respiratory Distress Syndrome/physiopathology , Respiratory Distress Syndrome/therapy , Respiratory Distress Syndrome/virology , Seasons , Single-Cell Analysis , Stromal Cells/metabolism
3.
PLoS Pathog ; 11(2): e1004642, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25668410

ABSTRACT

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8(+) cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K(b)PB(1703, D(b)PA(224), and D(b)NP(366) epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.


Subject(s)
Epitopes, T-Lymphocyte/immunology , Immunity, Heterologous/immunology , Immunodominant Epitopes/immunology , Immunologic Memory/immunology , Influenza A Virus, H7N9 Subtype/immunology , Orthomyxoviridae Infections/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Cross Reactions/immunology , Disease Models, Animal , Female , Flow Cytometry , Male , Mice , Mice, Inbred C57BL
4.
J Gen Virol ; 95(Pt 2): 350-362, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24243730

ABSTRACT

Type I alveolar epithelial cells are a replicative niche for influenza in vivo, yet their response to infection is not fully understood. To better characterize their cellular responses, we have created an immortalized murine lung epithelial type I cell line (LET1). These cells support spreading influenza virus infection in the absence of exogenous protease and thus permit simultaneous analysis of viral replication dynamics and host cell responses. LET1 cells can be productively infected with human, swine and mouse-adapted strains of influenza virus and exhibit expression of an antiviral transcriptional programme and robust cytokine secretion. We characterized influenza virus replication dynamics and host responses of lung type I epithelial cells and identified the capacity of epithelial cell-derived type I IFN to regulate specific modules of antiviral effectors to establish an effective antiviral state. Together, our results indicate that the type I epithelial cell can play a major role in restricting influenza virus infection without contribution from the haematopoietic compartment.


Subject(s)
Epithelial Cells/immunology , Epithelial Cells/virology , Immunity, Innate , Influenza A virus/immunology , Influenza A virus/physiology , Virus Replication , Animals , Cell Line , Interferon Type I/immunology , Interferon Type I/metabolism , Mice , Mice, Inbred C57BL
5.
Sci Data ; 1: 140033, 2014.
Article in English | MEDLINE | ID: mdl-25977790

ABSTRACT

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.


Subject(s)
Host-Pathogen Interactions , Influenza A virus , Influenza, Human/virology , Orthomyxoviridae Infections/virology , Animals , Data Collection , Databases, Factual , Humans , Influenza A virus/pathogenicity , Influenza A virus/physiology , Influenza, Human/physiopathology , Mice , Orthomyxoviridae Infections/physiopathology , Systems Biology
6.
PLoS One ; 8(9): e74863, 2013.
Article in English | MEDLINE | ID: mdl-24073225

ABSTRACT

Influenza viruses exhibit large, strain-dependent differences in pathogenicity in mammalian hosts. Although the characteristics of severe disease, including uncontrolled viral replication, infection of the lower airway, and highly inflammatory cytokine responses have been extensively documented, the specific virulence mechanisms that distinguish highly pathogenic strains remain elusive. In this study, we focused on the early events in influenza infection, measuring the growth rate of three strains of varying pathogenicity in the mouse airway epithelium and simultaneously examining the global host transcriptional response over the first 24 hours. Although all strains replicated equally rapidly over the first viral life-cycle, their growth rates in both lung and tracheal tissue strongly diverged at later times, resulting in nearly 10-fold differences in viral load by 24 hours following infection. We identified separate networks of genes in both the lung and tracheal tissues whose rapid up-regulation at early time points by specific strains correlated with a reduced viral replication rate of those strains. The set of early-induced genes in the lung that led to viral growth restriction is enriched for both NF-κB binding site motifs and members of the TREM1 and IL-17 signaling pathways, suggesting that rapid, NF-κB -mediated activation of these pathways may contribute to control of viral replication. Because influenza infection extending into the lung generally results in severe disease, early activation of these pathways may be one factor distinguishing high- and low-pathogenicity strains.


Subject(s)
Host-Pathogen Interactions , Lung/virology , Orthomyxoviridae Infections/virology , Orthomyxoviridae/physiology , Orthomyxoviridae/pathogenicity , Trachea/virology , Virus Replication/immunology , Animals , Biomarkers/metabolism , Cells, Cultured , Female , Gene Expression Profiling , Lung/immunology , Lung/metabolism , Mice , Mice, Inbred C57BL , NF-kappa B/genetics , NF-kappa B/metabolism , Oligonucleotide Array Sequence Analysis , Orthomyxoviridae/genetics , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/immunology , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , Signal Transduction , Trachea/immunology , Trachea/metabolism
7.
Methods Mol Biol ; 1031: 177-88, 2013.
Article in English | MEDLINE | ID: mdl-23824900

ABSTRACT

In vivo influenza infection models are critical for understanding viral dynamics and host responses during infection. Mouse models are extremely useful for infection studies requiring a high number of test animals. The vast array of gene knockout mice available is particularly helpful in investigating a particular gene's contributions to infection. Thus, more in vivo scientific experimentation of influenza has been done on mice than any other animal model. Here, we describe the technique of intranasal inoculation of mice and methods for assessing the severity of disease and humane endpoints, and discuss data acquired from infection of female C57BL/6J mice.


Subject(s)
Administration, Intranasal/methods , Influenza, Human/pathology , Influenza, Human/virology , Animals , Disease Models, Animal , Female , Humans , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza, Human/genetics , Mice , Mice, Knockout
8.
Am J Physiol Lung Cell Mol Physiol ; 304(7): L481-8, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23355384

ABSTRACT

During influenza virus infection, it is unclear how much alveolar cell loss can be tolerated before the host succumbs to the disease. We sought to define relevant correlates of disease severity in the mouse influenza model, hypothesizing that a susceptibility threshold exists for alveolar epithelial cell loss. We compared lung pathology, virus spread, alveolar epithelial cell depletion, arterial blood oxygenation, physiological responses measured by unrestrained plethysmography, and oxygen consumption and carbon dioxide production by gas analysis in mice at intervals after infection with virus strains and doses that cause mild (x31) or severe (PR/8) influenza. Both mild and severe infections showed similar degrees of lung damage and virus dissemination until day 6 after inoculation but diverged in survival outcomes from day 9. Day 6 PR/8-infected mice had normal respiratory and gas exchange functions with 10% type I cell loss. However, day 10 PR/8-infected mice had 40% type I cell loss with a concomitant drastic decreases in tidal and minute volumes, Vo(2), Vco(2), and arterial blood oxygenation, compared with a maximum 3% type I cell loss for x31 on day 10 when they recovered body weight and respiratory functions. Alterations in breaths per minute, expiratory time, and metabolic rate were observed in both infections. A threshold for maintenance of proper respiratory function appears to be crossed once 10% of alveolar type I cells are lost. These data indicate that lethality in influenza virus infection is a matter of degree rather than quality.


Subject(s)
Epithelial Cells/metabolism , Influenza A virus , Orthomyxoviridae Infections/metabolism , Oxygen Consumption , Pulmonary Alveoli/metabolism , Pulmonary Gas Exchange , Animals , Disease Models, Animal , Epithelial Cells/pathology , Epithelial Cells/virology , Female , Mice , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/physiopathology , Pulmonary Alveoli/pathology , Pulmonary Alveoli/physiopathology , Pulmonary Alveoli/virology , Respiratory Mechanics
9.
Cell Tissue Res ; 343(1): 13-21, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20848130

ABSTRACT

Infection by influenza virus leads to respiratory failure characterized by acute lung injury associated with alveolar edema, necrotizing bronchiolitis, and excessive bleeding. Severe reactions to infection that lead to hospitalizations and/or death are frequently attributed to an exuberant host response, with excessive inflammation and damage to the epithelial cells that mediate respiratory gas exchange. The respiratory mucosa serves as a physical and chemical barrier to infection, producing mucus and surfactants, anti-viral mediators, and inflammatory cytokines. The airway epithelial cell layer also serves as the first and overwhelmingly primary target for virus infection and growth. This review details immune events during influenza infection from the viewpoint of the epithelial cells, secretory host defense mechanisms, cell death, and recovery.


Subject(s)
Epithelial Cells/immunology , Epithelial Cells/virology , Immunity, Innate/immunology , Influenza, Human/immunology , Orthomyxoviridae/immunology , Respiratory System/cytology , Cell Death , Humans , Influenza, Human/virology , Respiratory System/immunology , Respiratory System/virology
10.
Eur J Immunol ; 39(2): 359-71, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19152336

ABSTRACT

The ability of TLR agonists to promote adaptive immune responses is attributed to their ability to robustly activate innate immunity. However, it has been observed that, for adjuvants in actual use in research and vaccination, TLR signaling is dispensable for generating humoral immunity. Here, we examined the role of TLR5 and MyD88 in promoting innate and humoral immunity to flagellin using a prime/boost immunization regimen. We observed that eliminating TLR5 greatly reduced flagellin-induced cytokine production, except for IL-18, and ablated DC maturation but did not significantly impact flagellin's ability to promote humoral immunity. Elimination of MyD88, which will ablate signaling through TLR and IL-1beta/IL-18 generated by Nod-like receptors, reduced, but did not eliminate flagellin's promotion of humoral immunity. In contrast, loss of the innate immune receptor for profilin-like protein (PLP), TLR11, greatly reduced the ability of PLP to elicit humoral immunity. Together, these results indicate that, firstly, the degree of innate immune activation induced by TLR agonists may be in great excess of that needed to promote humoral immunity and, secondly, there is considerable redundancy in mechanisms that promote the humoral immune response upon innate immune recognition of flagellin. Thus, it should be possible to design innate immune activators that are highly effective vaccine adjuvants yet avoid the adverse events associated with systemic TLR activation.


Subject(s)
Antibodies/blood , Dendritic Cells/immunology , Flagellin/immunology , Myeloid Differentiation Factor 88/immunology , Toll-Like Receptor 5/immunology , Animals , Antibody Formation , Cytokines/biosynthesis , Cytokines/immunology , Dendritic Cells/metabolism , Immunity, Active , Immunity, Innate , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Ovalbumin/immunology , Toll-Like Receptor 5/genetics , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism
11.
J Immunol ; 180(12): 8280-5, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18523294

ABSTRACT

Sudden exposure of human populations to chemicals, pathogens, or radiation has the potential to result in substantial morbidity. A potential means of rapidly protecting such populations might be to activate innate host defense pathways, which can provide broad protection against a variety of insults. However, innate immune activators can, by themselves, result in severe inflammatory pathology, which in large part is driven by hemopoietic-derived cytokines such as TNF-alpha. We reasoned that, because it preferentially activates epithelial cells, the TLR5 agonist flagellin might not induce severe inflammatory pathology and yet be an ideal agent to provide such non-specific protection, particularly at the mucosal surfaces that serve as a front line of host defense. In accordance, we observed that systemic treatment of mice with purified flagellin did not induce the serologic, histopathologic, and clinical hallmarks of inflammation that are induced by LPS but yet protected mice against chemicals, pathogens, and ionizing radiation. Flagellin-elicited radioprotection required TLR5, the TLR signaling adaptor MyD88, and was effective if given between 2 h before to 4 h after exposure to irradiation. Flagellin-elicited radioprotection was, in part, mediated via effects on cells in bone marrow but yet rescued mortality without a pronounced rescue of radiation-induced anemia or leukopenia. Thus, systemic administration of flagellin may be a relatively safe means of providing temporary non-specific protection against a variety of challenges.


Subject(s)
Dextran Sulfate/administration & dosage , Flagellin/administration & dosage , Gamma Rays , Radiation-Protective Agents/administration & dosage , Rotavirus Infections/prevention & control , Salmonella Infections, Animal/prevention & control , Administration, Oral , Animals , Cytokines/biosynthesis , Dextran Sulfate/adverse effects , Flagellin/therapeutic use , Gamma Rays/adverse effects , Immunity, Innate/drug effects , Immunity, Innate/radiation effects , Injections, Intraperitoneal , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/adverse effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/physiology , Radiation-Protective Agents/therapeutic use , Rotavirus Infections/immunology , Salmonella Infections, Animal/immunology , Toll-Like Receptor 5/deficiency , Toll-Like Receptor 5/genetics , Toll-Like Receptor 5/physiology
12.
J Immunol ; 180(11): 7184-92, 2008 Jun 01.
Article in English | MEDLINE | ID: mdl-18490717

ABSTRACT

The TLR5 agonist flagellin induces innate and adaptive immune responses in a MyD88-dependent manner and is under development as a vaccine adjuvant. In vitro studies indicate that, compared with other bacteria-derived adjuvants, flagellin is a very potent activator of proinflammatory gene expression and cytokine production from cells of nonhemopoietic origin. However, the role of nonhemopoietic cells in promoting flagellin-induced immune responses in vivo remains unclear. To investigate the relative contributions of the nonhemopoietic (radioresistant) and the hemopoietic (radiosensitive) compartments, we measured both innate and adaptive immune responses of flagellin-treated MyD88 radiation bone marrow chimeras. We observed that radiosensitive and radioresistant cells played distinct roles in the innate response to flagellin, with the radiosensitive cells producing the majority of the TNF-alpha, IL-12, and IL-6 cytokines and the radioresistant cells most of the KC, IP-10, and MCP-1 cytokines. Direct activation of either compartment alone by flagellin initiated dendritic cell costimulatory molecule up-regulation and induced a significant humoral immune response to the protein itself as well as to coinjected OVA. However, robust humoral responses were only observed when MyD88 was present in both cell compartments. Further studies revealed that hemopoietic and nonhemopoietic expression of the cytokines TNF-alpha and IL-6, but not IL-1, played an important role in promoting flagellin-induced Ab responses. Thus, in vivo both radioresistant and hemopoietic cells play key nonredundant roles in mediating innate and adaptive immune responses to flagellin.


Subject(s)
Cytokines/metabolism , Hematopoietic System/immunology , Immunity, Innate , Myeloid Differentiation Factor 88/metabolism , Toll-Like Receptor 5/metabolism , Animals , Antibody Formation , Cytokines/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Flagellin/immunology , Hematopoietic System/cytology , Hematopoietic System/metabolism , Immunity, Active , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/immunology , Radiation Chimera , Toll-Like Receptor 5/agonists , Toll-Like Receptor 5/immunology
13.
J Clin Invest ; 117(12): 3909-21, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18008007

ABSTRACT

Activation of TLRs by bacterial products results in rapid activation of genes encoding products designed to protect the host from perturbing microbes. In the intestine, which is colonized by a large and diverse population of commensal bacteria, TLR signaling may not function in a simple on/off mode. Here, we show that the flagellin receptor TLR5 has an essential and nonredundant role in protecting the gut from enteric microbes. Mice lacking TLR5 (TLR5KO mice) developed spontaneous colitis, as assessed by well-defined clinical, serologic, and histopathologic indicators of this disorder. Compared with WT littermates, TLR5KO mice that had not yet developed robust colitis exhibited decreased intestinal expression of TLR5-regulated host defense genes despite having an increased bacterial burden in the colon. In contrast, such TLR5KO mice displayed markedly increased colonic expression of hematopoietic-derived proinflammatory cytokines, suggesting that elevated levels of bacterial products may result in activation of other TLRs that drive colitis in TLR5KO mice. In accordance, deletion of TLR4 rescued the colitis of TLR5KO mice in that mice lacking both TLR4 and TLR5 also had elevated bacterial loads in the colon but lacked immunological, histopathological, and clinical evidence of colitis. That an engineered innate immune deficiency ultimately results in spontaneous intestinal inflammation supports the notion that an innate immune deficiency might underlie some instances of inflammatory bowel disease.


Subject(s)
Colitis/genetics , Gene Deletion , Immunity, Innate/genetics , Inflammatory Bowel Diseases/genetics , Toll-Like Receptor 5/genetics , Animals , Bacteria/immunology , Colitis/immunology , Colitis/microbiology , Colitis/pathology , Colon/immunology , Colon/pathology , Cytokines/genetics , Cytokines/immunology , Flagellin/immunology , Inflammation Mediators/immunology , Inflammatory Bowel Diseases/immunology , Inflammatory Bowel Diseases/microbiology , Inflammatory Bowel Diseases/pathology , Mice , Mice, Knockout , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Toll-Like Receptor 5/immunology
14.
J Immunol ; 177(5): 2810-8, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16920916

ABSTRACT

Bacterial flagellin, the primary structural component of flagella, is a dominant target of humoral immunity upon infection by enteric pathogens and in Crohn's disease. To better understand how such responses may be regulated, we sought to define, in mice, basic mechanisms that regulate generation of flagellin-specific Igs. We observed that, in response to i.p. injection with flagellin, generation of flagellin-specific Ig required activation of innate immunity in that these responses were ablated in MyD88-deficient mice and that flagellin from Helicobacter pylori, which is known not to activate TLR5, also did not elicit Abs. Mice lacking alphabeta T cells (TCRbeta(null)) were completely deficient in their ability to make flagellin Abs in various contexts indicating that, in contrast to common belief, generation of flagellin-specific Ig is absolutely T cell dependent. In contrast to Ab responses to whole flagella (H serotyping), responses to flagellin monomers displayed only moderate serospecificity. Whereas neither oral nor rectal administration of flagellin elicited a strong serum Ab response, induction of colitis with dextran sodium sulfate resulted in a MyD88-dependent serum Ab response to endogenous flagellin, suggesting that, in an inflammatory milieu, TLR signaling promotes acquisition of Abs to intestinal flagellin. Thus, acquisition of a humoral immune response to flagellin requires activation of innate immunity, is T cell dependent, and can originate from flagellin in the intestinal tract in inflammatory conditions in the intestine.


Subject(s)
Antibody Formation/immunology , Flagellin/immunology , Immunity, Innate/immunology , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antibodies/blood , Antibodies/immunology , Colitis/immunology , Cytokines/immunology , Flagellin/isolation & purification , Helicobacter pylori/immunology , Intestinal Mucosa/immunology , Kinetics , Mice , Mice, Transgenic , Myeloid Differentiation Factor 88
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