Julie L. McAuley

MUC1 cell surface mucin is a critical element of the mucosal barrier to infection

Cell surface mucin glycoproteins are highly expressed by all mucosal tissues, yet their physiological role is currently unknown. We hypothesized that cell surface mucins protect mucosal cells from infection. A rapid progressive increase in gastrointestinal expression of mucin 1 (Muc1) cell surface mucin followed infection of mice with the bacterial pathogen Campylobacter jejuni. In the first week following oral infection, C. jejuni was detected in the systemic organs of the vast majority of Muc1(-/-) mice but never in Muc1(+/+) mice. Although C. jejuni entered gastrointestinal epithelial cells of both Muc1(-/-) and Muc1(+/+) mice, small intestinal damage as manifested by increased apoptosis and enucleated and shed villous epithelium was more common in Muc1(-/-) mice. Using radiation chimeras, we determined that prevention of systemic infection in wild-type mice was due exclusively to epithelial Muc1 rather than Muc1 on hematopoietic cells. Expression of MUC1-enhanced resistance to C. jejuni cytolethal distending toxin (CDT) in vitro and CDT null C. jejuni showed lower gastric colonization in Muc1(-/-) mice in vivo. We believe this is the first in vivo experimental study to demonstrate that cell surface mucins are a critical component of mucosal defence and that the study provides the foundation for exploration of their contribution to epithelial infectious and inflammatory diseases.

Influenza enhances susceptibility to natural acquisition of and disease due to Streptococcus pneumoniae in ferrets.

The role of respiratory viruses in the transmission of Streptococcus pneumoniae is poorly understood. Key questions, such as which serotypes are most fit for transmission and disease and whether influenza virus alters these parameters in a serotype-specific manner, have not been adequately studied. In a novel model of transmission in ferrets, we demonstrated that pneumococcal transmission and disease were enhanced if donors had previously been infected with influenza virus. Bacterial titers in nasal wash, the incidence of mucosal and invasive disease, and the percentage of contacts that were infected all increased. In contact ferrets, viral infection increased their susceptibility to S. pneumoniae acquisition both in terms of the percentage infected and the distance over which they could acquire infection. These influenza-mediated effects on colonization, transmission, and disease were dependent on the pneumococcal strain. Overall, these data argue that the relationship between respiratory viral infections, acquisition of pneumococci, and development of disease in humans needs further study to be better understood.

PB1-F2 Proteins from H5N1 and 20th Century Pandemic Influenza Viruses Cause Immunopathology

With the recent emergence of a novel pandemic strain, there is presently intense interest in understanding the molecular signatures of virulence of influenza viruses. PB1-F2 proteins from epidemiologically important influenza A virus strains were studied to determine their function and contribution to virulence. Using 27-mer peptides derived from the C-terminal sequence of PB1-F2 and chimeric viruses engineered on a common background, we demonstrated that induction of cell death through PB1-F2 is dependent upon BAK/BAX mediated cytochrome c release from mitochondria. This function was specific for the PB1-F2 protein of A/Puerto Rico/8/34 and was not seen using PB1-F2 peptides derived from past pandemic strains. However, PB1-F2 proteins from the three pandemic strains of the 20th century and a highly pathogenic strain of the H5N1 subtype were shown to enhance the lung inflammatory response resulting in increased pathology. Recently circulating seasonal influenza A strains were not capable of this pro-inflammatory function, having lost the PB1-F2 proteins immunostimulatory activity through truncation or mutation during adaptation in humans. These data suggest that the PB1-F2 protein contributes to the virulence of pandemic strains when the PB1 gene segment is recently derived from the avian reservoir.

Influenza Virus Primes Mice for Pneumonia From Staphylococcus aureus

Superinfections from Staphylococcus aureus following influenza are an increasing concern. We assessed several laboratory and clinical strains in a mouse coinfection model with influenza virus. A methicillin-resistant USA300 clone and several recent clinical strains from patients with necrotizing pneumonia caused high mortality following influenza virus infection in mice. Both viral and bacterial lung titers were enhanced during coinfections compared with single infections. However, differences in titers did not correspond with differences in disease outcomes in a comparison of superinfections from a highly pathogenic strain with those from a poorly pathogenic strain. These strains did differ, however, in expression of Panton-Valentine leukocidin and in the degree of inflammatory lung damage each engendered. The viral cytotoxin PB1-F2 contributed to the negative outcomes. These data suggest that additional study of specific bacterial virulence factors involved in the pathogenesis of inflammation and lung damage during coinfections is needed.

Activation of the NLRP3 Inflammasome by IAV Virulence Protein PB1-F2 Contributes to Severe Pathophysiology and Disease

The ability for a host to recognize infection is critical for virus clearance and often begins with induction of inflammation. The PB1-F2 of pathogenic influenza A viruses (IAV) contributes to the pathophysiology of infection, although the mechanism for this is unclear. The NLRP3-inflammasome has been implicated in IAV pathogenesis, but whether IAV virulence proteins can be activators of the complex is unknown. We investigated whether PB1-F2-mediated activation of the NLRP3-inflammasome is a mechanism contributing to overt inflammatory responses to IAV infection. We show PB1-F2 induces secretion of pyrogenic cytokine IL-1β by activating the NLRP3-inflammasome, contributing to inflammation triggered by pathogenic IAV. Compared to infection with wild-type virus, mice infected with reverse engineered PB1-F2-deficient IAV resulted in decreased IL-1β secretion and cellular recruitment to the airways. Moreover, mice exposed to PB1-F2 peptide derived from pathogenic IAV had enhanced IL-1β secretion compared to mice exposed to peptide derived from seasonal IAV. Implicating the NLRP3-inflammasome complex specifically, we show PB1-F2 derived from pathogenic IAV induced IL-1β secretion was Caspase-1-dependent in human PBMCs and NLRP3-dependent in mice. Importantly, we demonstrate PB1-F2 is incorporated into the phagolysosomal compartment, and upon acidification, induces ASC speck formation. We also show that high molecular weight aggregated PB1-F2, rather than soluble PB1-F2, induces IL-1β secretion. Furthermore, NLRP3-deficient mice exposed to PB1-F2 peptide or infected with PB1-F2 expressing IAV were unable to efficiently induce the robust inflammatory response as observed in wild-type mice. In addition to viral pore forming toxins, ion channel proteins and RNA, we demonstrate inducers of NLRP3-inflammasome activation may include disordered viral proteins, as exemplified by PB1-F2, acting as host pathogen ‘danger’ signals. Elucidating immunostimulatory PB1-F2 mediation of NLRP3-inflammasome activation is a major step forward in our understanding of the aetiology of disease attributable to exuberant inflammatory responses to IAV infection.

The Effects of Influenza A Virus PB1-F2 Protein on Polymerase Activity Are Strain Specific and Do Not Impact Pathogenesis

ABSTRACT The influenza A virus PB1-F2 protein has been implicated as a virulence factor, but the mechanism by which it enhances pathogenicity is not understood. The PB1 gene segment of the H1N1 swine-origin influenza virus pandemic strain codes for a truncated PB1-F2 protein which terminates after 11 amino acids but could acquire the full-length form by mutation or reassortment. It is therefore important to understand the function and impact of this protein. We systematically assessed the effect that PB1-F2 expression has on viral polymerase activity, accumulation and localization of PB1, and replication in vitro and in mice. We used both the laboratory strain PR8 and a set of viruses engineered to study clinically relevant PB1-F2 proteins. PB1-F2 expression had modest effects on polymerase activity, PB1 accumulation, and replication that were cell type and virus strain dependent. Disruption of the PB1-F2 reading frame in a recent, seasonal H3N2 influenza virus strain did not affect these parameters, suggesting that this is not a universal function of the protein. Disruption of PB1-F2 expression in several backgrounds or expression of PB1-F2 from the 1918 pandemic strain or a 1956 H1N1 strain had no effect on viral lung loads in mice. Alternate mechanisms besides alterations to replication are likely responsible for the enhanced virulence in mammalian hosts attributed to PB1-F2 in previous studies.

Kinetics of Coinfection with Influenza A Virus and Streptococcus pneumoniae

Secondary bacterial infections are a leading cause of illness and death during epidemic and pandemic influenza. Experimental studies suggest a lethal synergism between influenza and certain bacteria, particularly Streptococcus pneumoniae, but the precise processes involved are unclear. To address the mechanisms and determine the influences of pathogen dose and strain on disease, we infected groups of mice with either the H1N1 subtype influenza A virus A/Puerto Rico/8/34 (PR8) or a version expressing the 1918 PB1-F2 protein (PR8-PB1-F2(1918)), followed seven days later with one of two S. pneumoniae strains, type 2 D39 or type 3 A66.1. We determined that, following bacterial infection, viral titers initially rebound and then decline slowly. Bacterial titers rapidly rise to high levels and remain elevated. We used a kinetic model to explore the coupled interactions and study the dominant controlling mechanisms. We hypothesize that viral titers rebound in the presence of bacteria due to enhanced viral release from infected cells, and that bacterial titers increase due to alveolar macrophage impairment. Dynamics are affected by initial bacterial dose but not by the expression of the influenza 1918 PB1-F2 protein. Our model provides a framework to investigate pathogen interaction during coinfections and to uncover dynamical differences based on inoculum size and strain.

Bacterial Sinusitis and Otitis Media following Influenza Virus Infection in Ferrets

ABSTRACT Streptococcus pneumoniae is the leading cause of otitis media, sinusitis, and pneumonia. Many of these infections result from antecedent influenza virus infections. In this study we sought to determine whether the frequency and character of secondary pneumococcal infections differed depending on the strain of influenza virus that preceded bacterial challenge. In young ferrets infected with influenza virus and then challenged with pneumococcus, influenza viruses of any subtype increased bacterial colonization of the nasopharynx. Nine out of 10 ferrets infected with H3N2 subtype influenza A viruses developed either sinusitis or otitis media, while only 1 out of 11 ferrets infected with either an H1N1 influenza A virus or an influenza B virus did so. These data may partially explain why bacterial complication rates are higher during seasons when H3N2 viruses predominate. This animal model will be useful for further study of the mechanisms that underlie viral-bacterial synergism.

Reduced mucin sulfonation and impaired intestinal barrier function in the hyposulfataemic NaS1 null mouse

Objective: Sulfate (SO42−) is an abundant component of intestinal mucins and its content is decreased in certain gastrointestinal diseases, including inflammatory bowel disease. In this study, the hyposulfataemic NaS1 sulfate transporter null (Nas1−/−) mice were used to investigate the physiological consequences of disturbed sulfate homeostasis on (1) intestinal sulfomucin content and mRNA expression; (2) intestinal permeability and proliferation; (3) dextran sulfate sodium (DSS)-induced colitis; and (4) intestinal barrier function against the bacterial pathogen, Campylobacter jejuni. Methods: Intestinal sulfomucins and sialomucins were detected by high iron diamine staining, permeability was assessed by fluorescein isothiocyanate (FITC)–dextran uptake, and proliferation was assessed by 5-bromodeoxyuridine (BrdU) incorporation. Nas1−/− and wild-type (Nas1+/+) mice received DSS in drinking water, and intestinal damage was assessed by histological, clinical and haematological measurements. Mice were orally inoculated with C jejuni, and intestinal and systemic infection was assessed. Ileal mRNA expression profiles of Nas1−/− and Nas1+/+ mice were determined by cDNA microarrays and validated by quantitative real-time PCR. Results: Nas1−/− mice exhibited reduced intestinal sulfomucin content, enhanced intestinal permeability and DSS-induced colitis, and developed systemic infections when challenged orally with C jejuni. The transcriptional profile of 41 genes was altered in Nas1−/− mice, with the most upregulated gene being pancreatic lipase-related protein 2 and the most downregulated gene being carbonic anhydrase 1 (Car1). Conclusion: Sulfate homeostasis is essential for maintaining a normal intestinal metabolic state, and hyposulfataemia leads to reduced intestinal sulfomucin content, enhanced susceptibility to toxin-induced colitis and impaired intestinal barrier to bacterial infection.

Immunopathogenic and Antibacterial Effects of H3N2 Influenza A Virus PB1-F2 Map to Amino Acid Residues 62, 75, 79, and 82

ABSTRACT The influenza A virus protein PB1-F2 has been linked to the pathogenesis of both primary viral and secondary bacterial infections. H3N2 viruses have historically expressed full-length PB1-F2 proteins with either proinflammatory (e.g., from influenza A/Hong Kong/1/1968 virus) or noninflammatory (e.g., from influenza A/Wuhan/359/1995 virus) properties. Using synthetic peptides derived from the active C-terminal portion of the PB1-F2 protein from those two viruses, we mapped the proinflammatory domain to amino acid residues L62, R75, R79, and L82 and then determined the role of that domain in H3N2 influenza virus pathogenicity. PB1-F2-derived peptides containing that proinflammatory motif caused significant morbidity, mortality, and pulmonary inflammation in mice, manifesting as increased acute lung injury and the presence of proinflammatory cytokines and inflammatory cells in the lungs compared to peptides lacking this motif, and better supported bacterial infection with Streptococcus pneumoniae. Infections of mice with an otherwise isogenic virus engineered to contain this proinflammatory sequence in PB1-F2 demonstrated increased morbidity resulting from primary viral infections and enhanced development of secondary bacterial pneumonia. The presence of the PB1-F2 noninflammatory (P62, H75, Q79, and S82) sequence in the wild-type virus mediated an antibacterial effect. These data suggest that loss of the inflammatory PB1-F2 phenotype that supports bacterial superinfection during adaptation of H3N2 viruses to humans, coupled with acquisition of antibacterial activity, contributes to the relatively diminished frequency of severe infections seen with seasonal H3N2 influenza viruses in recent decades compared to their first 2 decades of circulation.