【病毒外文文献】2014 Neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contr

Neutrophils Are Needed for an Effective Immune Response Against Pulmonary Rat Coronavirus g1005g3 Infection but also Contribute to Pathology g1006g3 g1007g3 Running Title Neutrophils in rat coronavirus infection g1008g3 Contents Category Animal Viruses Positive Strand RNA g1009g3 g1010g3 Anoria K Haick 1 2 Joanna P Rzepka 1 3 Elizabeth Brandon 1 Onesmo B Balemba 1 and Tanya g1011g3 A Miura 1 g1012g3 1 Department of Biological Sciences University of Idaho 875 Perimeter Dr MS 3051 Moscow g1013g3 ID 83844 3051 USA g1005g1004g3 2 Current Address Department of Obstetrics and Gynecology University of Washington School g1005g1005g3 of Medicine 1959 Northeast Pacific Street Seattle WA 98195 USA g1005g1006g3 3 Current Address Veterinary Medical Research and Development Incorporated 425 Northwest g1005g1007g3 Albion Drive Pullman WA 99163 USA g1005g1008g3 Corresponding Author Email tmiura uidaho edu Telephone 208 885 4940 Fax 208 885 g1005g1009g3 7905 g1005g1010g3 Word Count Summary 234 g1005g1011g3 Word Count Text 5499 g1005g1012g3 Tables 2 g1005g1013g3 Figures 6 g1006g1004g3 JGV Papers in Press Published December 9 2013 as doi 10 1099 vir 0 061986 0 g3 g1006g3 Summary g1006g1005g3 Polymorphonuclear neutrophils PMN infiltrate the respiratory tract early after viral g1006g1006g3 infection and can contribute to both host defense and pathology Coronaviruses are important g1006g1007g3 causes of respiratory tract infections ranging from mild to severe depending on the viral strain g1006g1008g3 This study evaluated the role of PMN during a non fatal pulmonary coronavirus infection in the g1006g1009g3 natural host Rat coronavirus RCoV causes respiratory disease in adult rats characterized by g1006g1010g3 an early PMN response viral replication and inflammatory lesions in the lungs mild weight loss g1006g1011g3 and effective resolution of infection To determine their role during RCoV infection PMN were g1006g1012g3 depleted and the effects on disease progression viral replication inflammatory response and g1006g1013g3 lung pathology were analyzed Compared to RCoV infection in control animals PMN depleted g1007g1004g3 rats had worsened disease with weight loss clinical signs mortality and prolonged pulmonary g1007g1005g3 viral replication PMN depleted animals had fewer macrophages and lymphocytes in the g1007g1006g3 respiratory tract corresponding with lower chemokine levels Combined with in vitro g1007g1007g3 experiments showing that PMN express cytokines and chemokines in response to RCoV infected g1007g1008g3 alveolar epithelial cells these findings support a role for PMN in eliciting an inflammatory g1007g1009g3 response to RCoV infection Despite their critical role in the protection from severe disease the g1007g1010g3 presence of PMN was correlated with hemorrhagic lesions epithelial barrier permeability and g1007g1011g3 cellular inflammation in the lungs This study demonstrated that while PMN are required for an g1007g1012g3 effective antiviral response they also contribute to lung pathology during RCoV infection g1007g1013g3 g1008g1004g3 g1008g1005g3 g1008g1006g3 g3 g1007g3 Introduction g1008g1007g3 Inflammatory responses triggered by respiratory viruses are necessary for the initiation of g1008g1008g3 effective antiviral immunity but can also become dysregulated and result in acute lung injury g1008g1009g3 and respiratory distress syndrome Polymorphonuclear neutrophils PMN infiltrate the airways g1008g1010g3 early after infection by respiratory viral pathogens including rhinoviruses influenza viruses g1008g1011g3 respiratory syncytial virus and coronaviruses The presence of PMN in the respiratory tract g1008g1012g3 during viral infection is frequently correlated with clinical symptoms or severe disease pathology g1008g1013g3 Bradley et al 2012 Denlinger et al 2011 Khanolkar et al 2009 McKean et al 2003 g1009g1004g3 Nagata et al 2008 Tumpey et al 2005 In contrast PMN have direct antiviral activities and g1009g1005g3 also function in the activation of innate and adaptive immune responses and thus can contribute g1009g1006g3 to effective antiviral responses Mantovani et al 2011 Tate et al 2012 Tate et al 2011 g1009g1007g3 Widegren et al 2011 Because PMN can be involved in both protective and pathologic immune g1009g1008g3 responses a complete understanding of their functions during viral infection may lead to the g1009g1009g3 design of therapeutic strategies that exploit the beneficial functions of PMN while limiting their g1009g1010g3 damaging effects in the lung g1009g1011g3 Coronaviruses CoV cause respiratory diseases in humans as well as companion and g1009g1012g3 agricultural animals Human CoV infections may result in mild common colds more serious g1009g1013g3 lower respiratory tract diseases or the highly fatal severe acute respiratory syndrome SARS or g1010g1004g3 Middle East Respiratory Syndrome MERS depending on the virus strain and the age and g1010g1005g3 immune status of the host Assiri et al 2013 Gaunt et al 2010 Lee et al 2003 PMN are g1010g1006g3 recruited to CoV infected tissues and either contribute to pathology or are necessary for an g1010g1007g3 effective immune response depending on the specific CoV and disease model The presence of g1010g1008g3 PMN corresponds to increased disease severity in humans and animals infected with SARS CoV g1010g1009g3 g3 g1008g3 or human CoV 229E Leong et al 2006 McKean et al 2003 Nagata et al 2008 Tsui et al g1010g1010g3 2003 During neurotropic murine coronavirus infection PMN contribute to brain pathology g1010g1011g3 Iacono et al 2006 but are also critical for the effective resolution of infection by promoting g1010g1012g3 blood brain barrier permeability which is needed for effective T cell recruitment to the brain g1010g1013g3 Hosking et al 2009 Zhou et al 2003 Despite these findings and the fact that CoVs g1011g1004g3 commonly infect the respiratory tract the functions of PMN during respiratory CoV infections g1011g1005g3 are not well understood g1011g1006g3 Rodent models of respiratory coronavirus infection are available for SARS CoV but not g1011g1007g3 the more common and milder CoV that circulate in human populations worldwide We have g1011g1008g3 developed a rat coronavirus RCoV model to determine the mechanisms that promote effective g1011g1009g3 resolution of a non fatal coronavirus infection in the lung RCoV is a natural pathogen of rats g1011g1010g3 that replicates and causes mild disease in the upper and lower respiratory tracts Funk et al g1011g1011g3 2009 Wojcinski Li et al 2007 Ofulue Sir et al 2000 Snipes g1013g1010g3 et al 1995 In agreement with these studies PMN effectively and specifically depleted PMN g1013g1011g3 from the blood of rats for at least 4 days followed by re population by day 6 post infection Fig g1013g1012g3 1b Importantly PMN serum did not reduce the numbers of other white blood cell types in g1013g1013g3 RCoV infected or uninfected animals Fig 1b and Supplemental Fig 1 Thus PMN is an g1005g1004g1004g3 effective specific tool for transient depletion of circulating PMN in rats g1005g1004g1005g3 PMN depleted and NRS treated animals were inoculated with RCoV and weighed and g1005g1004g1006g3 observed daily for clinical signs and mortality In agreement with our previous study RCoV g1005g1004g1007g3 infection of NRS treated rats did not result in mortality Funk et al 2009 In contrast treatment g1005g1004g1008g3 with PMN resulted in 28 mortality of RCoV infected rats by day 6 Fig 1c Of the 18 rats in g1005g1004g1009g3 the PMN RCoV group 1 succumbed to infection on day 2 and 4 others were humanely g1005g1004g1010g3 euthanized due to excessive weight loss and severe disease None of the mock infected animals g1005g1004g1011g3 either with or without PMN treatment died or required euthanasia during the course of the g1005g1004g1012g3 experiment All of the treatment groups exhibited weight loss early in the study and except for g1005g1004g1013g3 g3 g1010g3 the PMN RCoV group steadily regained weight beginning on day 3 Fig 1d In contrast g1005g1005g1004g3 PMN depleted rats that were infected with RCoV had steady weight loss through day 4 which g1005g1005g1005g3 remained low through day 8 Fig 1d Clinical scores were calculated daily as described in g1005g1005g1006g3 materials and methods Fig 1e NRS treated rats infected with RCoV showed no or only minor g1005g1005g1007g3 clinical signs during infection In contrast RCoV infection of PMN treated rats resulted in g1005g1005g1008g3 multiple clinical signs including hunched posture ruffled fur swollen face and neck bloody eye g1005g1005g1009g3 and nasal discharge and lethargy Therefore these rats had significantly increased mean clinical g1005g1005g1010g3 scores between days 1 and 8 post infection Fig 1e Surviving animals 72 had lower clinical g1005g1005g1011g3 scores after day 4 but did not return to complete health by day 8 The increased morbidity and g1005g1005g1012g3 mortality in rats treated with PMN which specifically depletes PMN from the bloodstream g1005g1005g1013g3 suggests that PMN are needed for protection against severe disease during RCoV infection g1005g1006g1004g3 g1005g1006g1005g3 PMN treatment reduces PMN recruitment and prolongs viral replication in the lungs g1005g1006g1006g3 To confirm that treatment with PMN inhibits recruitment of PMN to the respiratory g1005g1006g1007g3 tract PMN were quantified in bronchoalveolar lavage fluid BALF on days 4 8 and 12 post g1005g1006g1008g3 infection As expected from our previous study Funk et al 2009 PMN numbers increased in g1005g1006g1009g3 NRS RCoV treated rats by day 4 and declined to less than 5 by day 8 Fig 2a In rats treated g1005g1006g1010g3 with PMN PMN numbers in the BALF did not increase upon RCoV infection and remained g1005g1006g1011g3 low through day 12 despite their repopulation of the blood by day 6 Fig 1b To determine g1005g1006g1012g3 whether PMN are needed for clearance of RCoV viral titers from lung homogenates were g1005g1006g1013g3 compared in NRS and PMN treated rats Both groups had high levels of RCoV on day 4 g1005g1007g1004g3 which remained high in PMN treated rats through day 12 Fig 2b In contrast NRS treated g1005g1007g1005g3 g3 g1011g3 rats cleared the virus by day 8 post infection Thus recruitment of PMN to the respiratory tract g1005g1007g1006g3 correlated with effective clearance of RCoV from the lungs g1005g1007g1007g3 g1005g1007g1008g3 PMN are needed early during RCoV infection to protect against disease g1005g1007g1009g3 PMN are observed in the respiratory tract early after infection with RCoV Funk et al g1005g1007g1010g3 2009 but it is not known if their presence early during infection is important to later disease g1005g1007g1011g3 outcomes To establish transient depletion of PMN early during RCoV infection rats were g1005g1007g1012g3 treated with PMN serum one day before and two days after RCoV inoculation Fig 3a No g1005g1007g1013g3 PMN were detected in the BALF of PMN treated rats on day 4 post infection followed by g1005g1008g1004g3 recruitment of PMN to the respiratory tract by day 8 Fig 3b Despite the influx of PMN into g1005g1008g1005g3 the airways viral titers in the lungs remained high on day 8 Fig 3c suggesting that the g1005g1008g1006g3 presence of PMN alone is not sufficient to clear virus late in infection Transient PMN depletion g1005g1008g1007g3 resulted in 50 mortality by day 8 after RCoV infection and significant weight loss compared to g1005g1008g1008g3 NRS treated animals Fig 3d and e NRS RCoV rats initially lost weight which they re gained g1005g1008g1009g3 after day 3 Fig 3e Of the 6 animals that died during the study 1 succumbed to infection on g1005g1008g1010g3 day 3 and 5 were euthanized due to more than 20 weight loss and severe disease Clinical signs g1005g1008g1011g3 were apparent on days 2 8 post infection and were identical to those seen in rats given PMN g1005g1008g1012g3 throughout infection Data not shown These findings demonstrate that delayed recruitment of g1005g1008g1013g3 PMN to the lungs cannot compensate for their absence early during RCoV infection g1005g1009g1004g3 g3g1005g1009g1005g3 PMN promote pulmonary cellular infiltration during RCoV infection g1005g1009g1006g3 g3 g1012g3 The results of transient PMN depletion suggested that PMN are needed early during g1005g1009g1007g3 RCoV infection to limit disease severity and that later recruitment of PMN does not reduce viral g1005g1009g1008g3 titers Therefore we hypothesize that PMN have an indirect role in the effective response against g1005g1009g1009g3 RCoV infection RCoV infection induces cellular infiltration into the alveolar spaces Funk et g1005g1009g1010g3 al 2009 To determine whether PMN are required for cellular inflammation histological g1005g1009g1011g3 analysis of lung tissues was performed on PMN depleted and NRS treated rats during RCoV g1005g1009g1012g3 infection Focal areas of pneumonitis with PMN macrophages and lymphocytes were present in g1005g1009g1013g3 the lung sections from NRS treated but not PMN treated animals on day 4 post infection Fig g1005g1010g1004g3 4a Inflammatory lesions in the lungs of NRS treated animals were mostly localized in areas g1005g1010g1005g3 surrounding the bronchioles top panels Quantitative analysis of density indices of PMN g1005g1010g1006g3 macrophages and lymphocytes was performed on tissue sections from three animals per group g1005g1010g1007g3 PMN treatment significantly reduced the numbers of macrophages and lymphocytes in the g1005g1010g1008g3 lungs of RCoV infected animals Fig 4b corresponding with cell counts in BALF samples Fig g1005g1010g1009g3 4c To determine whether CD4 or CD8 positive lymphocytes were specifically reduced these g1005g1010g1010g3 cells were quantified in BALF by flow cytometry This analysis demonstrated a reduction in both g1005g1010g1011g3 CD4 and CD8 positive cells in the airways of PMN treated animals compared to NRS treated g1005g1010g1012g3 animals upon RCoV infection Fig 4d These data suggest that PMN are critical for the g1005g1010g1013g3 development of a cellular response to pulmonary RCoV infection g1005g1011g1004g3 g1005g1011g1005g3 i1PMN treated rats have reduced chemokine concentrations in the BALF during RCoV g1005g1011g1006g3 infection g1005g1011g1007g3 g3 g1013g3 The histology data demonstrated that PMN promote pulmonary cellular infiltration g1005g1011g1008g3 during RCoV infection and our previous studies showed RCoV induced chemokine expression g1005g1011g1009g3 Miura et al 2007 Funk et al 2009 To determine whether PMN contribute to this response g1005g1011g1010g3 we quantified chemokines in the BALF of PMN and NRS treated rats during RCoV infection g1005g1011g1011g3 Compared to mock inoculated animals RCoV infection increased levels of PMN specific g1005g1011g1012g3 chemokines CXCL 1 and CXCL 3 in NRS treated animals by day 4 which returned to mock g1005g1011g1013g3 levels by day 8 Fig 5a In contrast PMN depleted rats had significantly reduced levels of g1005g1012g1004g3 CXCL 1 and CXCL 3 in the BALF which corresponded with the lack of PMN recruitment to g1005g1012g1005g3 the lungs of depleted animals even after PMN had repopulated the blood Fig 1b and 2a Two g1005g1012g1006g3 additional chemokines that are induced by RCoV infection Funk et al 2009 interferon g1005g1012g1007g3 inducible protein 10 IP 10 CXCL 10 and monocyte chemoattractant protein 1 MCP 1 CCL 2 g1005g1012g1008g3 were quantified in BALF from PMN and NRS treated rats on day 4 post infection Fig 5b g1005g1012g1009g3 Both chemokines were induced by RCoV infection in NRS treated but not PMN treated rats g1005g1012g1010g3 suggesting that PMN are needed for chemokine production g1005g1012g1011g3 g1005g1012g1012g3 Proinflammatory response of PMN to RCoV infected alveolar epithelial cells in vitro g1005g1012g1013g3 Based on the data above we hypothesize that PMN recruited to the airways of RCoV g1005g1013g1004g3 infected rats produce cytokines and chemokines including CXCL 1 CXCL 3 IP 10 and CCL g1005g1013g1005g3 2 Type I alveolar epithelial AT1 cells are the primary cell type infected by RCoV within the g1005g1013g1006g3 distal lung Funk et al 2009 Furthermore RCoV infected AT1 like cells direct PMN functions g1005g1013g1007g3 in vitro Rzepka et al 2012 g3To determine whether RCoV infected AT1 cells direct expression g1005g1013g1008g3 of cytokines and chemokines by PMN we incubated PMN isolated from rat bone marrow in g1005g1013g1009g3 g3 g1005g1004 conditioned medium from RCoV infected RCoV AT1 or mock infected mock AT1 AT1 like g1005g1013g1010g3 cells The mRNA levels of 84 cytokines and chemokines were measured from PMN using g1005g1013g1011g3 quantitative RT PCR arrays Table 1 PMN that were incubated in RCoV AT1 medium had g1005g1013g1012g3 higher mRNA levels of proinflammatory cytokines IL 18 IL 1 IL 1 and TNF CXC g1005g1013g1013g3 chemokines CXCL 1 CXCL 2 IP 10 CXCL 11 and CC chemokines CCL 2 CCL 4 CCL g1006g1004g1004g3 7 CCL 9 CCL 12 and CCL 22 in comparison to PMN incubated in mock AT1 medium These g1006g1004g1005g3 findings demonstrated that PMN express proinflammatory cytokines and chemokines when g1006g1004g1006g3 exposed to RCoV infected epithelial cells This is in agreement with the reduced concentrations g1006g1004g1007g3 of chemokines in the BALF and cellular infiltration in the lungs of rats treated with PMN g1006g1004g1008g3 antibody compared to NRS treated rats during RCoV infection g1006g1004g1009g3 g1006g1004g1010g3 The presence of PMN in the lungs is associated with tissue damage g1006g1004g1011g3 Hemorrhagic lesions are observed on the surface of the lungs following the same kinetics g1006g1004g1012g3 as PMN recruitment during RCoV infection Funk et al 2009 Therefore we determined g1006g1004g1013g3 whether the presence of PMN correlated with visible lesions on the surface of rat lungs RCoV g1006g1005g1004g3 infection of NRS treated rats resulted in gross pulmonary lesions in all animals that were g1006g1005g1005g3 analyzed on day 4 and the majority of NRS treated animals did not have lesions on day 8 The g1006g1005g1006g3 presence of lesions on the lungs of NRS treated animals corresponded to increased numbers of g1006g1005g1007g3 PMN in the airways Table 2 In contrast none of the rats treated with PMN throughout g1006g1005g1008g3 infection had visible lesions which corresponded to the absence of PMN in the BALF Likewise g1006g1005g1009g3 in rats that received PMN only early during infection through day 2 no lesions were visible g1006g1005g1010g3 on the lungs on day 4 when PMN were also not detected in the BALF Late influx of PMN to g1006g1005g1011g3 g3 g1005g1005 the respiratory tracts of transiently depleted rats by day 8 corresponded to the presence of g1006g1005g1012g3 pulmonary lesions in 5 of the 6 animals evaluated In addition to surface lesions we measured g1006g1005g1013g3 total protein in the BALF of rats as an indicator of damage to the epithelial barrier When PMN g1006g1006g1004g3 were present in the BALF there was a corresponding increase in protein concentration Table 2 g1006g1006g1005g3 Furthermore animals that had low numbers of PMN also had low total protein concentrations in g1006g1006g1006g3 the BALF Collectively these findings implicate PMN in causing tissue injury including gross g1006g1006g1007g3 hemorrhagic lesions and epithelial permeability g1006g1006g1008g3 Histological analysis was performed to compare lung pathology in PMN depleted and g1006g1006g1009g3 NRS treated rats during RCoV infection Corresponding with focal areas of pneumonitis Fig g1006g1006g1010g3 4a NRS RCoV rats had severe bronchiolar and peribronchiolar inflammation necrosis and g1006g1006g1011g3 epithelial sloughing Fig 6b compared to mock inoculated rats Fig 6a Some alveoli of g1006g1006g1012g3 NRS RCoV rats contained transudate fluid dead cells and inflammatory cells Fig 6e g1006g1006g1013g3 compared to the clear alveoli of uninfected rats Fig 6d Interest