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1.  Role of CCL5 (RANTES) in Viral Lung Disease 
Journal of Virology  2006;80(16):8151-8157.
CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+ and CD8+ cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.
doi:10.1128/JVI.00496-06
PMCID: PMC1563837  PMID: 16873271
2.  The Chemokine MIP1α/CCL3 Determines Pathology in Primary RSV Infection by Regulating the Balance of T Cell Populations in the Murine Lung 
PLoS ONE  2010;5(2):e9381.
Background
CD8 T cells assist in the clearance of respiratory syncytial virus (RSV) infection from the lungs. However, disease after RSV infection is in part caused by excessive T cell activity, and a balance is therefore needed between beneficial and harmful cellular immune responses. The chemokine CCL3 (MIP1α) is produced following RSV infection and is broadly chemotactic for both T cells and natural killer (NK) cells. We therefore investigated its role in RSV disease.
Methodology/Principal Findings
CCL3 was produced biphasically, in both the early (day 1) and late (day 6–7) stages of infection. CCL3 depletion did not alter the recruitment of natural killer (NK) cells to the lungs during the early stage, but depletion did affect the later adaptive phase. While fewer T cells were recruited to the lungs of either CCL3 knockout or anti-CCL3 treated RSV infected mice, more RSV-specific pro-inflammatory T cells were recruited to the lung when CCL3 responses were impaired. This increase in RSV-specific pro-inflammatory T cells was accompanied by increased weight loss and illness after RSV infection.
Conclusions/Significance
CCL3 regulates the balance of T cell populations in the lung and can alter the outcome of RSV infection. Understanding the role of inflammatory mediators in the recruitment of pathogenic T cells to the lungs may lead to novel methods to control RSV disease.
doi:10.1371/journal.pone.0009381
PMCID: PMC2827540  PMID: 20195359
3.  The Number of RSV-Specific Memory CD8 T Cells in the Lung is Critical for their Ability to Inhibit Respiratory Syncytial Virus (RSV) Vaccine-Enhanced Pulmonary Eosinophilia1 
Children that were administered a formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine experienced enhanced respiratory disease, including pulmonary eosinophilia, after contracting a natural RSV infection. RSV vaccine-enhanced disease can be mimicked in BALB/c mice immunized with either FI-RSV or with a recombinant vaccinia virus (vacv) expressing the RSV attachment (G) protein. We have recently demonstrated that memory CD8 T cells directed against the RSV immunodominant M282−90 epitope inhibit the development of pulmonary eosinophilia in either vacvG- or FI-RSV-immunized mice by reducing the total number of Th2 cells in the lung after RSV challenge. Here we show that memory CD8 T cells specific to a subdominant epitope within the RSV fusion (F) protein fail to inhibit the development of pulmonary eosinophilia after RSV challenge of mice previously co-immunized with vacvF and with either vacvG or FI-RSV. We observed that the inability of RSV F85-specific memory CD8 T cells to inhibit the development of pulmonary eosinophilia was largely due to an inadequate total number of F85-specific memory CD8 T cells in the lung at early times after RSV challenge. Increasing the number of F85-specific memory CD8 T cells after immunization grants them the ability to inhibit RSV vaccine-enhanced pulmonary eosinophilia. Moreover, we demonstrate that RSV-specific memory CD8 T cells, when present in sufficient numbers, inhibit the production of the Th2-associated chemokines CCL17 and CCL22. Taken together, these results indicate that RSV-specific memory CD8 T cells may alter the trafficking of Th2 cells and eosinophils into the lung.
PMCID: PMC2587004  PMID: 19017987
Cytotoxic T cells; Th1/Th2 T cells; Eosinophils; Lung; Vaccination
4.  Pulmonary eosinophils and their role in immunopathologic responses to formalin-inactivated pneumonia virus of mice 
Enhanced disease is the term used to describe the aberrant Th2 skewed responses to naturally-acquired human respiratory syncytial virus (hRSV) infection observed in individuals vaccinated with formalin-inactivated viral antigens. Here we explore this paradigm with pneumonia virus of mice (PVM), a pathogen that faithfully reproduces features of severe hRSV infection in a rodent host. We demonstrate that PVM infection in mice vaccinated with formalin-inactivated antigens from PVM-infected cells (PVM Ags) yields Th2-skewed hypersensitivity, analogous to that observed in response to hRSV. Specifically, we detect elevated levels of IL-4, IL-5, IL-13, and eosinophils in bronchoalveolar lavage (BAL) fluid of PVM-infected mice that were vaccinated with PVM Ags, but not among mice vaccinated with formalin-inactivated antigens from uninfected cells (Ctrl Ags). Interestingly, infection in PVM Ag-vaccinated mice was associated with a ~10-fold reduction in lung virus titer and protection against weight loss when compared to infected mice vaccinated with Ctrl Ags, despite the absence of serum neutralizing antibodies. Given recent findings documenting a role for eosinophils in promoting clearance of hRSV in vivo, we explored the role of eosinophils in altering the pathogenesis of disease with eosinophil-deficient mice. We found that eosinophil deficiency had no impact on virus titer in PVM Ags-vaccinated mice, nor on weight loss or levels of CCL11 (eotaxin-1), interferon-γ, interleukin (IL)-5, or IL-13 in BAL fluid. However, levels of both IL-4 and CCL3 (macrophage inflammatory protein-1α) in BAL fluid were markedly diminished in PVM Ag-vaccinated, PVM-infected eosinophil-deficient mice when compared to wild type controls (246 words).
doi:10.4049/jimmunol.0802270
PMCID: PMC2754814  PMID: 19542471
eosinophils; cytokines; inflammation; viral infection; lung
5.  Differential Role for TLR3 in Respiratory Syncytial Virus-Induced Chemokine Expression 
Journal of Virology  2005;79(6):3350-3357.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young infants worldwide. Previous studies have reported that the induction of interleukin-8/CXCL8 and RANTES/CCL5 correlates with disease severity in humans. The production of these chemokines is elicited by viral replication and is NF-κB dependent. RSV, a negative-sense single-stranded RNA virus, requires full-length positive-sense RNA for synthesis of new viral RNA. The aim of our studies was to investigate whether active viral replication by RSV could evoke chemokine production through TLR3-mediated signaling pathways. In TLR3-transfected HEK 293 cells, live RSV preferentially activated chemokines in both a time- and dose-dependent manner compared to vector controls. RSV was also shown to upregulate TLR3 in human lung fibroblasts and epithelial cells (MRC-5 and A549). Targeting the expression of TLR3 with small interfering RNA decreased synthesis of IP-10/CXCL10 and CCL5 but did not significantly reduce levels of CXCL8. Blocking the expression of the adapter protein MyD88 established a role for MyD88 in CXCL8 production, whereas CCL5 synthesis was found to be MyD88 independent. Production of CCL5 by RSV was induced directly through TLR3 signaling pathways and did not require interferon (IFN) signaling through the IFN-α/β receptor. TLR3 did not affect viral replication, since equivalent viral loads were recovered from RSV-infected cells despite altered TLR3 expression. Taken together, our studies indicate that TLR3 mediates inflammatory cytokine and chemokine production in RSV-infected epithelial cells.
doi:10.1128/JVI.79.6.3350-3357.2005
PMCID: PMC1075725  PMID: 15731229
6.  Differential Chemokine Expression following Respiratory Virus Infection Reflects Th1- or Th2-Biased Immunopathology 
Journal of Virology  2006;80(9):4521-4527.
Respiratory syncytial virus (RSV) is a major viral pathogen of infants that also reinfects adults. During RSV infection, inflammatory host cell recruitment to the lung plays a central role in determining disease outcome. Chemokines mediate cell recruitment to sites of inflammation and are influenced by, and influence, the production of cytokines. We therefore compared chemokine production in a mouse model of immunopathogenic RSV infection in which either Th1 or Th2 immunopathology is induced by prior sensitization to individual RSV proteins. Chemokine expression profiles were profoundly affected by the nature of the pulmonary immunopathology: “Th2” immunopathology in BALB/c mice was associated with increased and prolonged expression of CCL2 (MCP-1), CXCL10 (IP-10), and CCL11 (eotaxin) starting within 24 h of challenge. C57BL/6 mice with “Th2” pathology (enabled by a deficiency of CD8+ cells) also showed increased CCL2 production. No differences in chemokine receptor expression were detected. Chemokine blockers may therefore be of use for children with bronchiolitis.
doi:10.1128/JVI.80.9.4521-4527.2006
PMCID: PMC1472012  PMID: 16611912
7.  Immunoprotectivity of HLA-A2 CTL Peptides Derived from Respiratory Syncytial Virus Fusion Protein in HLA-A2 Transgenic Mouse 
PLoS ONE  2011;6(9):e25500.
Identification of HLA-restricted CD8+ T cell epitopes is important to study RSV-induced immunity and illness. We algorithmically analyzed the sequence of the fusion protein (F) of respiratory syncytial virus (RSV) and generated synthetic peptides that can potentially bind to HLA-A*0201. Four out of the twenty-five 9-mer peptides tested: peptides 3 (F33–41), 13 (F214–222), 14 (F273–281), and 23 (F559–567), were found to bind to HLA-A*0201 with moderate to high affinity and were capable of inducing IFN-γ and IL-2 secretion in lymphocytes from HLA-A*0201 transgenic (HLA-Tg) mice pre-immunized with RSV or recombinant adenovirus expressing RSV F. HLA-Tg mice were immunized with these four peptides and were found to induce both Th1 and CD8+ T cell responses in in vitro secondary recall. Effector responses induced by these peptides were observed to confer differential protection against live RSV challenge. These peptides also caused better recovery of body weight loss induced by RSV. A significant reduction of lung viral load was observed in mice immunized with peptide 23, which appeared to enhance the levels of inflammatory chemokines (CCL17, CCL22, and IL-18) but did not increase eosinophil infiltration in the lungs. Whereas, significant reduction of infiltrated eosinophils induced by RSV infection was found in mice pre-immunized with peptide 13. Our results suggest that HLA-A2-restricted epitopes of RSV F protein could be useful for the development of epitope-based RSV vaccine.
doi:10.1371/journal.pone.0025500
PMCID: PMC3183052  PMID: 21980478
8.  Cell-Specific Expression of RANTES, MCP-1, and MIP-1α by Lower Airway Epithelial Cells and Eosinophils Infected with Respiratory Syncytial Virus 
Journal of Virology  1998;72(6):4756-4764.
Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infancy, a syndrome characterized by wheezing, respiratory distress, and the pathologic findings of peribronchial mononuclear cell infiltration and release of inflammatory mediators by basophil and eosinophil leukocytes. Composition and activation of this cellular response are thought to rely on the discrete target cell selectivity of C-C chemokines. We demonstrate that infection in vitro of human epithelial cells of the lower respiratory tract by RSV induced dose- and time-dependent increases in mRNA and protein secretion for RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α). Production of MCP-1 and MIP-1α was selectively localized only in epithelial cells of the small airways and lung. Exposure of epithelial cells to gamma interferon (IFN-γ), in combination with RSV infection, induced a significant increase in RANTES production that was synergistic with respect to that obtained by RSV infection or IFN-γ treatment alone. Epithelial cell-derived chemokines exhibited a strong chemotactic activity for normal human blood eosinophils. Furthermore, eosinophils were susceptible to RSV and released RANTES and MIP-1α as a result of infection. Therefore, the inflammatory process in RSV-induced bronchiolitis appears to be triggered by the infection of epithelial cells and further amplified via mechanisms driven by IFN-γ and by the secretion of eosinophil chemokines.
PMCID: PMC110009  PMID: 9573240
9.  Whole Blood Gene Expression Profiles to Assess Pathogenesis and Disease Severity in Infants with Respiratory Syncytial Virus Infection 
PLoS Medicine  2013;10(11):e1001549.
In this study, Mejias and colleagues found that specific blood RNA profiles of infants with RSV LRTI allowed for specific diagnosis, better understanding of disease pathogenesis, and better assessment of disease severity.
Please see later in the article for the Editors' Summary
Background
Respiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity.
Methods and Findings
This was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n = 16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%–98%]) and specificity (98% [95% CI 88%–99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants (<6 mo). We identified a genomic score that significantly correlated with outcomes of care including a clinical disease severity score and, more importantly, length of hospitalization and duration of supplemental O2.
Conclusions
Blood RNA profiles of infants with RSV LRTI allow specific diagnosis, better understanding of disease pathogenesis, and assessment of disease severity. This study opens new avenues for biomarker discovery and identification of potential therapeutic or preventive targets, and demonstrates that large microarray datasets can be translated into a biologically meaningful context and applied to the clinical setting.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lower respiratory tract infections (LRTIs)—bacterial and viral infections of the lungs and airways (the tubes that take oxygen-rich air to the lungs)—are major causes of illness and death in children worldwide. Pneumonia (infection of the lungs) alone is responsible for 14% of all child deaths. The leading cause of viral LTRIs in children is respiratory syncytial virus (RSV), which is readily transmitted from person to person by direct contact with nasal fluids or airborne droplets. Almost all children have an RSV infection before their second birthday, but most have only minor symptoms similar to those of a common cold and are cared for at home. Unfortunately, some children develop more serious conditions when they become infected with RSV, such as pneumonia or bronchiolitis (swelling and mucus build-up in the bronchioles, the smallest air passages in the lungs). These children have to be admitted to the hospital for supportive care—there is no specific treatment for RSV infection—such as the provision of supplemental oxygen.
Why Was This Study Done?
The lack of a treatment (and of a vaccine) for RSV is largely due to our incomplete understanding of the cellular events and reactions, including the host immune response, that occur during the development of an RSV infection (disease pathogenesis). Moreover, based on physical examination and available diagnostic tools, it is impossible to predict which children infected with RSV will develop a serious condition that requires hospitalization and which ones can be safely nursed at home. Here, the researchers use microarrays to analyze the global host response to acute RSV LTRI in infants, to define gene expression patterns that are specific to RSV infection rather than infection with other common respiratory viruses, and to identify biomarkers that indicate the severity of RSV infection. “Microarray” analysis allows researchers to examine gene expression patterns (“RNA transcriptional profiles”) in, for example, whole blood; a biomarker is a molecule whose level in bodily fluids or tissues indicates how a disease might develop and helps with patient classification.
What Did the Researchers Do and Find?
The researchers compared the RNA transcriptional profile in whole blood taken from children less than two years old hospitalized with RSV, human rhinovirus, or influenza virus infection (rhinovirus and influenza are two additional viral causes of LRTI), and from healthy infants. Using “statistical group comparisons,” they identified more than 2,000 transcripts that were differentially expressed in blood from 45 infants with RSV infection and from 14 healthy matched controls. Genes related to interferon function (interferons are released by host cells in response to the presence of disease-causing organisms) and neutrophil function (neutrophils are immune system cells that, like interferons, are involved in the innate immune response, the body's first line of defense against infection) were among the most overexpressed genes in infants infected with RSV. Genes regulating T and B cells (components of the adaptive immune response, the body's second-line of defense against infection) were among the most underexpressed genes. This specific transcriptional profile, which was validated in three additional groups of infants, accurately distinguished between infants infected with RSV and those infected with human rhinovirus or influenza virus. Finally, a “molecular distance to health” score (a numerical score that quantifies the transcriptional perturbation associated with an illness) was correlated with the clinical disease severity score of the study participants, with how long they needed supplemental oxygen, and with their duration of hospitalization.
What Do These Findings Mean?
These findings suggest that it might be possible to use whole blood RNA transcriptional profiles to distinguish between infants infected with RSV and those with other viruses that commonly cause LRTI. Moreover, if these findings can be replicated in more patients (including non-hospitalized children), gene expression profiling might provide a strategy for triaging patients with RSV infections when they first present to an emergency department and for monitoring clinical changes during the course of the infection, particularly given the development of molecular tools that might soon enable the “real time” acquisition of transcriptional profiles in the clinical setting. Finally, although certain aspects of the study design limit the accuracy and generalizability of the study's findings, these data provide new insights into the pathogenesis of RSV infection and open new avenues for the discovery of biomarkers for RSV infection and for the identification of therapeutic and preventative targets.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001549.
This study is further discussed in a PLOS Medicine Perspective by Peter Openshaw
The US Centers for Disease Control and Prevention provides information about RSV infection
The US National Heart, Lung, and Blood Institute provides information about the respiratory system and about RSV infections
The UK National Health Service Choices website provides information about bronchiolitis
The British Lung Foundation also provides information on RSV and on bronchiolitis
MedlinePlus provides links to other resources about RSV infections and about pneumonia (in English and Spanish); the MedlinePlus encyclopedia has a page on bronchiolitis (in English and Spanish)
PATH is an international non-profit organization investigating new RSV vaccines
doi:10.1371/journal.pmed.1001549
PMCID: PMC3825655  PMID: 24265599
10.  Pseudotyped Adeno-Associated Virus 2/9-Delivered CCL11 shRNA Alleviates Lung Inflammation in an Allergen-Sensitized Mouse Model 
Human Gene Therapy  2012;23(11):1156-1165.
Abstract
Airway infiltration by eosinophils is a major characteristic of chronic asthma. CCL11 (eotaxin-1) is secreted by lung epithelial cells and functions as the major chemokine for eosinophil recruitment. Pseudotyped adeno-associated virus (AAV) 2/9, composed by the AAV2 rep and AAV9 cap genes, can efficiently target lung epithelial cells and might carry gene sequences with therapeutic potential for asthma. This study aimed to determine whether pseudotyped AAV2/9 virus carrying the small hairpin RNA targeting CCL11 and expressed by CMV/U6 promoter could reduce eosinophilia and asthmatic responses in mite allergen-sensitized mice. Mice were sensitized by intraperitoneal and challenged by intratracheal injection with recombinant Dermatophagoides pteronyssinus group 2 allergen (rDp2). AAV2/9 viral vectors were intratracheally injected three days before the first challenge. AAV2/9 sh47 virus significantly reduced airway hyperresponsiveness, airway resistance, CCL11 levels, and eosinophilia in the lungs of sensitized mice. Th2 cytokines, including interleukins (IL)-4, IL-5, and IL-10, were also significantly reduced in the bronchoalveolar lavage fluid of AAV2/9 sh47 virus-treated mice. Th2 cytokine levels were also reduced in rDp2-stimulated mediastinal lymphocytes in treated mice. However, serum levels of rDp2-specific IgG1 and IgE, as well as Th2 cytokine levels in rDp2-stimulated splenocyte culture supernatants, were comparable to the sensitized control group. The results suggest that AAV2/9 sh47 virus relieved local instead of systemic inflammatory responses. Therefore, the CMV/U6 promoter with AAV2/9 viral vector, which is preferable to target lung epithelia cells, might be applied as a novel therapeutic approach for asthma.
Wu and colleagues show that intratracheal injection of AAV2/9 vector encoding short hairpin RNA (shRNA) against CCL11 (AAV2/9 sh47) leads to a significant reduction of airway hyperresponsiveness and airway resistance in the lungs of allergen-sensitized mice. Serum levels of allergen-specific IgG1 and IgE, as well as helper T cell type 2 cytokine levels, were similar in the AAV2/9 and sensitized control groups, suggesting that this approach relieves local but not systemic inflammatory responses.
doi:10.1089/hum.2012.012
PMCID: PMC3498884  PMID: 22913580
11.  Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2 ▿  
Journal of Virology  2008;83(3):1492-1500.
Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.
doi:10.1128/JVI.00671-08
PMCID: PMC2620898  PMID: 19019963
12.  Early phase resolution of mucosal eosinophilic inflammation in allergic rhinitis 
Respiratory Research  2010;11(1):54.
Background
It is widely assumed that apoptosis of eosinophils is a central component of resolution of allergic airway disease. However, this has not been demonstrated in human allergic airways in vivo. Based on animal in vivo observations we hypothesised that steroid-induced resolution of human airway eosinophilic inflammation involves inhibition of CCL5 (RANTES), a CC-chemokine regulating eosinophil and lymphocyte traffic, and elimination of eosinophils without evident occurrence of apoptotic eosinophils in the diseased tissue.
Objective
To determine mucosal eosinophilia, apoptotic eosinophils, general cell apoptosis and cell proliferation, and expression of CCL5 and CCL11 (eotaxin) in human allergic airway tissues in vivo at resolution of established symptomatic eosinophilic inflammation.
Methods
Twenty-one patients with intermittent (birch and/or grass) allergic rhinitis received daily nasal allergen challenges for two seven days' periods separated by more than two weeks washout. Five days into these "artificial pollen seasons", nasal treatment with budesonide was instituted and continued for six days in a double blinded, randomized, placebo-controlled, and crossover design. This report is a parallel group comparison of nasal biopsy histochemistry data obtained on the final day of the second treatment period.
Results
Treatments were instituted when clinical rhinitis symptoms had been established. Compared to placebo, budesonide reduced tissue eosinophilia, and subepithelial more than epithelial eosinophilia. Steroid treatment also attenuated tissue expression of CCL5, but CCL11 was not reduced. General tissue cell apoptosis and epithelial cell proliferation were reduced by budesonide. However, apoptotic eosinophils were not detected in any biopsies, irrespective of treatment.
Conclusions
Inhibition of CCL5-dependent recruitment of cells to diseased airway tissue, and reduced cell proliferation, reduced general cell apoptosis, but not increased eosinophil apoptosis, are involved in early phase steroid-induced resolution of human allergic rhinitis.
doi:10.1186/1465-9921-11-54
PMCID: PMC2873933  PMID: 20459697
13.  Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection 
BMC Immunology  2014;15(1):41.
Background
Respiratory syncytial virus (RSV) infects the lung epithelium where it stimulates the production of numerous host cytokines that are associated with disease burden and acute lung injury. Characterizing the host cytokine response to RSV infection, the regulation of host cytokines and the impact of neutralizing an RSV-inducible cytokine during infection were undertaken in this study.
Methods
A549, primary human small airway epithelial (SAE) cells and wild-type, TIR-domain-containing adapter-inducing interferon-β (Trif) and mitochondrial antiviral-signaling protein (Mavs) knockout (KO) mice were infected with RSV and cytokine responses were investigated by ELISA, multiplex analysis and qPCR. Neutralizing anti-leukemia inhibitory factor (LIF) IgG or control IgG was administered to a group of wild-type animals prior to RSV infection.
Results and discussion
RSV-infected A549 and SAE cells release a network of cytokines, including newly identified RSV-inducible cytokines LIF, migration inhibitory factor (MIF), stem cell factor (SCF), CCL27, CXCL12 and stem cell growth factor beta (SCGF-β). These RSV-inducible cytokines were also observed in the airways of mice during an infection. To identify the regulation of RSV inducible cytokines, Mavs and Trif deficient animals were infected with RSV. In vivo induction of airway IL-1β, IL-4, IL-5, IL-6, IL-12(p40), IFN-γ, CCL2, CCL5, CCL3, CXCL1, IP-10/CXCL10, IL-22, MIG/CXCL9 and MIF were dependent on Mavs expression in mice. Loss of Trif expression in mice altered the RSV induction of IL-1β, IL-5, CXCL12, MIF, LIF, CXCL12 and IFN-γ. Silencing of retinoic acid–inducible gene-1 (RIG-I) expression in A549 cells had a greater impact on RSV-inducible cytokines than melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and Trif expression. To evaluate the role of LIF in the airways during RSV infection, animals were treated with neutralizing anti-LIF IgG, which enhanced RSV pathology observed with increased airspace protein content, apoptosis and airway hyperresponsiveness compared to control IgG treatment.
Conclusions
RSV infection in the epithelium induces a network of immune factors to counter infection, primarily in a RIG-I dependent manner. Expression of LIF protects the lung from lung injury and enhanced pathology during RSV infection.
Electronic supplementary material
The online version of this article (doi:10.1186/s12865-014-0041-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12865-014-0041-4
PMCID: PMC4189665  PMID: 25277705
Respiratory syncytial virus; Immune response; Pathogen recognition receptors; Gene expression
14.  SUBVERSION OF PULMONARY DENDRITIC CELL FUNCTION BY PARAMYXOVIRUS INFECTIONS1 
Lower respiratory tract infections caused by the paramyxoviruses human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are characterized by short-lasting virus-specific immunity and often long term airway morbidity, both of which may be the result of alterations in the antigen presenting function of the lung which follow these infections. In this study, we investigated whether hMPV and RSV experimental infections alter the phenotype and function of dendritic cells (DC) subsets which are recruited to the lung. Characterization of lung DC trafficking demonstrated a differential recruitment of plasmacytoid DC (pDC), conventional DC (cDC) and interferon-producing killer DC (IKDC) to the lung and draining lymph nodes after hMPV and RSV infection. In vitro infection of lung DC indicated that in pDC, production of IFN-α, TNF-α, and CCL5 was induced only by hMPV while CCL3 and CCL4 were induced by both viruses. In cDC, a similar repertoire of cytokines was induced by hMPV and RSV, except for IFN-β, which was not induced by RSV. The function of lung pDC was altered following hMPV or RSV infection in vivo, as we demonstrated a reduced capacity of lung pDC to produce IFN-α as well as other cytokines including IL-6, TNF-α, CCL2, CCL3 and CCL4 in response to TLR9 agonist. Moreover, we observed an impaired capacity of cDC from infected mice to present Ag to CD4+ T cells, an effect that lasted beyond the acute phase of infection. Our findings suggest that acute paramyxovirus infections can alter the long term immune function of pulmonary DC.
doi:10.4049/jimmunol.0802262
PMCID: PMC2865244  PMID: 19234204
Dendritic cells; Lung; Viral; Cytokines; Cell trafficking
15.  Strain-specific requirement for eosinophils in the recruitment of T cells to the lung during the development of allergic asthma 
The Journal of Experimental Medicine  2008;205(6):1285-1292.
Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 ΔdblGATA mice (eosinophil-null mice via the ΔDblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.
doi:10.1084/jem.20071836
PMCID: PMC2413027  PMID: 18490489
16.  CCL26-Targeted siRNA Treatment of Alveolar Type II Cells Decreases Expression of CCR3-Binding Chemokines and Reduces Eosinophil Migration: Implications in Asthma Therapy 
The underlying inflammation present in chronic airway diseases is orchestrated by increased expression of CC chemokines that selectively recruit leukocyte populations into the pulmonary system. Human CCL26 signals through CC chemokine receptor 3 (CCR3), is dramatically upregulated in challenged asthmatics, and stimulates recruitment of eosinophils (EOSs) and other leukocytes. CCL26 participates in regulation of its receptor CCR3 and modulates expression of a variety of chemokines in alveolar type II cells. Utilizing the A549 alveolar type II epithelial cell culture model, we carried out studies to test the hypothesis that CCL26-siRNA treatment of these cells would ameliorate Th2-driven release of the eotaxins and other CCR3 ligands that would, in turn, decrease recruitment and activation of EOSs. Results demonstrate that CCL26-siRNA treatments decreased interleukin-4-induced CCL26 and CCL24 expression by >70%. CCL26-directed small-interfering RNA (siRNA) treatments significantly decreased release of CCL5 (RANTES), CCL15 (MIP-1δ), CCL8 (MCP-2), and CCL13 (MCP-4). In bioactivity assays it was shown that EOS migration and activation were reduced up to 80% and 90%, respectively, when exposed to supernatants of CCL26-siRNA-treated cells. These results provide evidence that CCL26 may be an appropriate target for development of new therapeutic agents designed to alleviate the underlying inflammation associated with chronic diseases of the airways.
doi:10.1089/jir.2008.0051
PMCID: PMC2941665  PMID: 19203252
17.  Development of Safe and Effective RSV Vaccine by Modified CD4 Epitope in G Protein Core Fragment (Gcf) 
PLoS ONE  2014;9(4):e94269.
Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in infants and young children worldwide, but currently no safe and effective vaccine is available. The RSV G glycoprotein (RSVG), a major attachment protein, is an important target for the induction of protective immune responses during RSV infection. However, it has been thought that a CD4+ T cell epitope (a.a. 183–195) within RSVG is associated with pathogenic pulmonary eosinophilia. To develop safe and effective RSV vaccine using RSV G protein core fragment (Gcf), several Gcf variants resulting from modification to CD4+ T cell epitope were constructed. Mice were immunized with each variant Gcf, and the levels of RSV-specific serum IgG were measured. At day 4 post-challenge with RSV subtype A or B, lung viral titers and pulmonary eosinophilia were determined and changes in body weight were monitored. With wild type Gcf derived from RSV A2 (wtAGcf), although RSV A subtype-specific immune responses were induced, vaccine-enhanced disease characterized by excessive pulmonary eosinophil recruitment and body weight loss were evident, whereas wtGcf from RSV B1 (wtBGcf) induced RSV B subtype-specific immune responses without the signs of vaccine-enhanced disease. Mice immunized with Th-mGcf, a fusion protein consisting CD4+ T cell epitope from RSV F (F51–66) conjugated to mGcf that contains alanine substitutions at a.a. position 185 and 188, showed higher levels of RSV-specific IgG response than mice immunized with mGcf. Both wtAGcf and Th-mGcf provided complete protection against RSV A2 and partial protection against RSV B. Importantly, mice immunized with Th-mGcf did not develop vaccine-enhanced disease following RSV challenge. Immunization of Th-mGcf provided protection against RSV infection without the symptom of vaccine-enhanced disease. Our study provides a novel strategy to develop a safe and effective mucosal RSV vaccine by manipulating the CD4+ T cell epitope within RSV G protein.
doi:10.1371/journal.pone.0094269
PMCID: PMC3988050  PMID: 24736750
18.  Human Eosinophils Express Functional CCR7 
Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5–primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM–loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5–primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5–primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7.
doi:10.1165/rcmb.2012-0499OC
PMCID: PMC3727873  PMID: 23449735
basic immunology; chemokines; eosinophils
19.  Respiratory Syncytial Virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice 
Respiratory Research  2010;11(1):125.
Background
Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity.
Methods
Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood.
Results
RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes.
Conclusions
RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.
doi:10.1186/1465-9921-11-125
PMCID: PMC2946301  PMID: 20843364
20.  CCL3 and Viral Chemokine-Binding Protein gG Modulate Pulmonary Inflammation and Virus Replication during Equine Herpesvirus 1 Infection▿  
Journal of Virology  2007;82(4):1714-1722.
CCL3 is a proinflammatory chemokine that mediates many of the cellular changes occurring in pulmonary disease. Here, CCL3−/− mice were used to investigate the role of this chemokine during respiratory herpesvirus infection. Compared to wild-type mice, CCL3−/− mice infected with the alphaherpesvirus equine herpesvirus 1 (EHV-1) displayed reduced body weight loss but had higher pulmonary viral loads. Lungs from infected CCL3−/− mice suffered a milder interstitial pneumonia, and fewer immune cells were recovered from the pulmonary airways after infection. We could also demonstrate that herpesvirus-encoded chemokine-binding glycoprotein G (gG) was capable of inhibiting the chemotactic functions of CCL3. This CCL3-mediated chemotaxis, however, was restored in the presence of gG-specific antibodies, which puts into question the advertised use of gG deletion mutants as marker vaccines. In summary, we concluded that CCL3 is a major player in controlling herpesvirus replication in the target organ, the lung, and does so by evoking a strong inflammatory response. The immunomodulatory activity of CCL3 is balanced by the expression of viral gG, whose chemokine-binding activity is mitigated in secondary infections by the production of anti-gG antibodies.
doi:10.1128/JVI.02137-07
PMCID: PMC2258710  PMID: 18077722
21.  Vβ14+ T Cells Mediate the Vaccine-Enhanced Disease Induced by Immunization with Respiratory Syncytial Virus (RSV) G Glycoprotein but Not with Formalin-Inactivated RSV 
Journal of Virology  2004;78(16):8753-8760.
Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4+ T cells expressing Vβ14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vβ14+ T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vβ14 component of the TCR. vvGs-immunized mice treated with anti-Vβ14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vβ14+ T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vβ chain usage confirmed a bias toward Vβ14 expression on CD4+ T cells from vvGs-immunized mice, whereas the CD4+ T cells in FI-RSV-immunized mice expressed a diverse array of Vβ chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.
doi:10.1128/JVI.78.16.8753-8760.2004
PMCID: PMC479099  PMID: 15280483
22.  Murine lung eosinophil activation and chemokine production in allergic airway inflammation 
Cellular & molecular immunology  2010;7(5):361-374.
Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.
doi:10.1038/cmi.2010.31
PMCID: PMC3045045  PMID: 20622891
allergy; chemokines; eosinophils; lung; mouse
23.  Murine lung eosinophil activation and chemokine production in allergic airway inflammation 
Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.
doi:10.1038/cmi.2010.31
PMCID: PMC3045045  PMID: 20622891
allergy; chemokines; eosinophils; lung; mouse
24.  Association between Respiratory Syncytial Virus Activity and Pneumococcal Disease in Infants: A Time Series Analysis of US Hospitalization Data 
PLoS Medicine  2015;12(1):e1001776.
Daniel Weinberger and colleagues examine a possible interaction between two serious respiratory infections in children under 2 years of age.
Please see later in the article for the Editors' Summary
Background
The importance of bacterial infections following respiratory syncytial virus (RSV) remains unclear. We evaluated whether variations in RSV epidemic timing and magnitude are associated with variations in pneumococcal disease epidemics and whether changes in pneumococcal disease following the introduction of seven-valent pneumococcal conjugate vaccine (PCV7) were associated with changes in the rate of hospitalizations coded as RSV.
Methods and Findings
We used data from the State Inpatient Databases (Agency for Healthcare Research and Quality), including >700,000 RSV hospitalizations and >16,000 pneumococcal pneumonia hospitalizations in 36 states (1992/1993–2008/2009). Harmonic regression was used to estimate the timing of the average seasonal peak of RSV, pneumococcal pneumonia, and pneumococcal septicemia. We then estimated the association between the incidence of pneumococcal disease in children and the activity of RSV and influenza (where there is a well-established association) using Poisson regression models that controlled for shared seasonal variations. Finally, we estimated changes in the rate of hospitalizations coded as RSV following the introduction of PCV7. RSV and pneumococcal pneumonia shared a distinctive spatiotemporal pattern (correlation of peak timing: ρ = 0.70, 95% CI: 0.45, 0.84). RSV was associated with a significant increase in the incidence of pneumococcal pneumonia in children aged <1 y (attributable percent [AP]: 20.3%, 95% CI: 17.4%, 25.1%) and among children aged 1–2 y (AP: 10.1%, 95% CI: 7.6%, 13.9%). Influenza was also associated with an increase in pneumococcal pneumonia among children aged 1–2 y (AP: 3.2%, 95% CI: 1.7%, 4.7%). Finally, we observed a significant decline in RSV-coded hospitalizations in children aged <1 y following PCV7 introduction (−18.0%, 95% CI: −22.6%, −13.1%, for 2004/2005–2008/2009 versus 1997/1998–1999/2000). This study used aggregated hospitalization data, and studies with individual-level, laboratory-confirmed data could help to confirm these findings.
Conclusions
These analyses provide evidence for an interaction between RSV and pneumococcal pneumonia. Future work should evaluate whether treatment for secondary bacterial infections could be considered for pneumonia cases even if a child tests positive for RSV.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Respiratory infections—bacterial and viral infections of the lungs and the airways (the tubes that take oxygen-rich air to the lungs)—are major causes of illness and death in children worldwide. Pneumonia (infection of the lungs) alone is responsible for about 15% of all child deaths. The leading cause of bacterial pneumonia in children is Streptococcus pneumoniae, which is transmitted through contact with infected respiratory secretions. S. pneumoniae usually causes noninvasive diseases such as bronchitis, but sometimes the bacteria invade the lungs, the bloodstream, or the covering of the brain, where they cause pneumonia, septicemia, or meningitis, respectively. These potentially fatal invasive pneumococcal diseases can be treated with antibiotics but can also be prevented by vaccination with pneumococcal conjugate vaccines such as PCV7. The leading cause of viral pneumonia is respiratory syncytial virus (RSV), which is also readily transmitted through contact with infected respiratory secretions. Almost all children have an RSV infection before their second birthday—RSV usually causes a mild cold-like illness. However, some children infected with RSV develop pneumonia and have to be admitted to hospital for supportive care such as the provision of supplemental oxygen; there is no specific treatment for RSV infection.
Why Was This Study Done?
Co-infections with bacteria and viruses can sometimes have a synergistic effect and lead to more severe disease than an infection with either type of pathogen (disease-causing organism) alone. For example, influenza infections increase the risk of invasive pneumococcal disease. But does pneumococcal disease also interact with RSV infection? It is important to understand the interaction between pneumococcal disease and RSV to improve the treatment of respiratory infections in young children, but the importance of bacterial infections following RSV infection is currently unclear. Here, the researchers undertake a time series analysis of US hospitalization data to investigate the association between RSV activity and pneumococcal disease in infants. Time series analysis uses statistical methods to analyze data collected at successive, evenly spaced time points.
What Did the Researchers Do and Find?
For their analysis, the researchers used data collected between 1992/1993 and 2008/2009 by the State Inpatient Databases on more than 700,000 hospitalizations for RSV and more than 16,000 hospitalizations for pneumococcal pneumonia or septicemia among children under two years old in 36 US states. Using a statistical technique called harmonic regression to measure seasonal variations in disease incidence (the rate of occurrence of new cases of a disease), the researchers show that RSV and pneumococcal pneumonia shared a distinctive spatiotemporal pattern over the study period. Next, using Poisson regression models (another type of statistical analysis), they show that RSV was associated with significant increases (increases unlikely to have happened by chance) in the incidence of pneumococcal disease. Among children under one year old, 20.3% of pneumococcal pneumonia cases were associated with RSV activity; among children 1–2 years old, 10.1% of pneumococcal pneumonia cases were associated with RSV activity. Finally, the researchers report that following the introduction of routine vaccination in the US against S. pneumoniae with PCV7 in 2000, there was a significant decline in hospitalizations for RSV among children under one year old.
What Do These Findings Mean?
These findings provide evidence for an interaction between RSV and pneumococcal pneumonia and indicate that RSV is associated with increases in the incidence of pneumococcal pneumonia, particularly in young infants. Notably, the finding that RSV hospitalizations declined after the introduction of routine pneumococcal vaccination suggests that some RSV hospitalizations may have a joint viral–bacterial etiology (cause), although it is possible that PCV7 vaccination reduced the diagnosis of RSV because fewer children were hospitalized with pneumococcal disease and subsequently tested for RSV. Because this is an ecological study (an observational investigation that looks at risk factors and outcomes in temporally and geographically defined populations), these findings do not provide evidence for a causal link between hospitalizations for RSV and pneumococcal pneumonia. The similar spatiotemporal patterns for the two infections might reflect another unknown factor shared by the children who were hospitalized for RSV or pneumococcal pneumonia. Moreover, because pooled hospitalization discharge data were used in this study, these results need to be confirmed through analysis of individual-level, laboratory-confirmed data. Importantly, however, these findings support the initiation of studies to determine whether treatment for bacterial infections should be considered for children with pneumonia even if they have tested positive for RSV.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001776.
The US National Heart, Lung, and Blood Institute provides information about the respiratory system and about pneumonia
The US Centers for Disease Control and Prevention provides information on all aspects of pneumococcal disease and pneumococcal vaccination, including personal stories and information about RSV infection
The UK National Health Service Choices website provides information about pneumonia (including a personal story) and about pneumococcal diseases
KidsHealth, a website provided by the US-based non-profit Nemours Foundation, includes information on pneumonia and on RSV (in English and Spanish)
MedlinePlus provides links to other resources about pneumonia, RSV infections, and pneumococcal infections (in English and Spanish)
HCUPnet provides aggregated hospitalization data from the State Inpatient Databases used in this study
doi:10.1371/journal.pmed.1001776
PMCID: PMC4285401  PMID: 25562317
25.  Rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages 
Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated ovalbumin-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared to sham-infected, ovalbumin-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13 and IFN-γ. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemistry showed eotaxin-1 in the lung macrophages of virus-infected, ovalbumin-treated mice, and confocal fluorescence microscopy revealed co-localization of rhinovirus, eotaxin-1 and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from ovalbumin-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from ovalbumin-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2 and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from ovalbumin-sensitized and -challenged mice reduced eosinophilic inflammation and airway hyperreactivity following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.
doi:10.4049/jimmunol.1000286
PMCID: PMC3208235  PMID: 20644177

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