During viral infection, inflammation and recovery are tightly controlled by competing proinflammatory and regulatory immune pathways. Respiratory syncytial virus (RSV) is the leading global cause of infantile bronchiolitis, which is associated with recurrent wheeze and asthma diagnosis in later life. Th2-driven disease has been well described under some conditions for RSV-infected mice. In the present studies, we used the Foxp3DTR mice (which allow specific conditional depletion of Foxp3+ T cells) to investigate the functional effects of regulatory T cells (Tregs) during A2-strain RSV infection. Infected Treg-depleted mice lost significantly more weight than wild-type mice, indicating enhanced disease. This enhancement was characterized by increased cellularity in the bronchoalveolar lavage (BAL) fluid and notable lung eosinophilia not seen in control mice. This was accompanied by abundant CD4+ and CD8+ T cells exhibiting an activated phenotype and induction of interleukin 13 (IL-13)- and GATA3-expressing Th2-type CD4+ T cells that remained present in the airways even 14 days after infection. Therefore, Treg cells perform vital anti-inflammatory functions during RSV infection, suppressing pathogenic T cell responses and inhibiting lung eosinophilia. These findings provide additional evidence that dysregulation of normal immune responses to viral infection may contribute to severe RSV disease.
Peter Openshaw discusses the challenges in advancing respiratory syncytial virus (RSV) treatments and the implications of a study by Mejias and colleagues using a newly identified gene signature for diagnosis and prediction of RSV severity.
Please see later in the article for the Editors' Summary
During severe influenza pandemics healthcare demand can exceed clinical capacity to provide normal standards of care. Community Assessment Tools (CATs) could provide a framework for triage decisions for hospital referral and admission. CATs have been developed based on evidence that supports the recognition of severe influenza and pneumonia in the community (including resource limited settings) for adults, children and infants, and serious feverish illness in children. CATs use six objective criteria and one subjective criterion, any one or more of which should prompt urgent referral and admission to hospital. A retrospective evaluation of the ability of CATs to predict use of hospital-based interventions and patient outcomes in a pandemic was made using the first recorded routine clinical assessment on or shortly after admission from 1520 unselected patients (800 female, 480 children <16 years) admitted with PCR confirmed A(H1N1)pdm09 infection (the FLU-CIN cohort). Outcome measures included: any use of supplemental oxygen; mechanical ventilation; intravenous antibiotics; length of stay; intensive or high dependency care; death; and “severe outcome” (combined: use of intensive or high dependency care or death during admission). Unadjusted and multivariable analyses were conducted for children (age <16 years) and adults. Each CATs criterion independently identified both use of clinical interventions that would in normal circumstances only be provided in hospital and patient outcome measures. “Peripheral oxygen saturation ≤92% breathing air, or being on oxygen” performed well in predicting use of resources and outcomes for both adults and children; supporting routine measurement of peripheral oxygen saturation when assessing severity of disease. In multivariable analyses the single subjective criterion in CATs “other cause for clinical concern” independently predicted death in children and in adults predicted length of stay, mechanical ventilation and “severe outcome”; supporting the role of clinical acumen as an important independent predictor of serious illness.
Although generally mild, the 2009–2010 influenza A/H1N1 pandemic caused two major surges in hospital admissions in the UK. The characteristics of patients admitted during successive waves are described.
Data were systematically obtained on 1520 patients admitted to 75 UK hospitals between May 2009 and January 2010. Multivariable analyses identified factors predictive of severe outcome.
Patients aged 5–54 years were over-represented compared with winter seasonal admissions for acute respiratory infection, as were non-white ethnic groups (first wave only). In the second wave patients were less likely to be school age than in the first wave, but their condition was more likely to be severe on presentation to hospital and they were more likely to have delayed admission. Overall, 45% had comorbid conditions, 16.5% required high dependency (level 2) or critical (level 3) care and 5.3% died. As in 1918–1919, the likelihood of severe outcome by age followed a W-shaped distribution. Pre-admission antiviral drug use decreased from 13.3% to 10% between the first and second waves (p=0.048), while antibiotic prescribing increased from 13.6% to 21.6% (p<0.001). Independent predictors of severe outcome were age 55–64 years, chronic lung disease (non-asthma, non-chronic obstructive pulmonary disease), neurological disease, recorded obesity, delayed admission (≥5 days after illness onset), pneumonia, C-reactive protein ≥100 mg/litre, and the need for supplemental oxygen or intravenous fluid replacement on admission.
There were demographic, ethnic and clinical differences between patients admitted with pandemic H1N1 infection and those hospitalised during seasonal influenza activity. Despite national policies favouring use of antiviral drugs, few patients received these before admission and many were given antibiotics.
Influenza; human; influenza A virus; H1N1 subtype; hospitalisation; mortality; critical care; clinical epidemiology; pneumonia; tobacco and the lung; bronchoscopy; paediatric asthma; paediatric lung disaese; paediatric physician; respiratory infection; viral infection; bacterial infection; asthma; cytokine biology; lymphocyte biology
Respiratory syncytial virus (RSV) is a major cause of bronchiolitis in infants. It is also responsible for high morbidity and mortality in the elderly. Programmed death ligands (PD-Ls) on antigen-presenting cells interact with receptors on T cells to regulate immune responses. The programmed death receptor-ligand 1/programmed death receptor 1 (PD-L1-PD-1) pathway is inhibitory in chronic viral infections, but its role in acute viral infections is unclear. We hypothesized that bronchial epithelial cell (BEC) expression of PD-Ls would inhibit local effector CD8+ T cell function. We report that RSV infection of primary human BECs strongly induces PD-L1 expression. In a co-culture system of BECs with purified CD8+ T cells, we demonstrated that RSV-infected BECs increased CD8+ T cell activation, proliferation, and antiviral function. Blocking PD-L1 on RSV-infected BECs co-cultured with CD8+ T cells enhanced CD8+ T cell IFN-γ, IL-2, and granzyme B production. It also decreased the virus load of the BECs. Based on our findings, we believe therapeutic strategies that target the PD-L1-PD-1 pathway might increase antiviral immune responses to RSV and other acute virus infections.
To determine clinical characteristics of patients hospitalized in the United Kingdom with pandemic (H1N1) 2009, we studied 1,520 patients in 75 National Health Service hospitals. We characterized patients who acquired influenza nosocomially during the pandemic (H1N1) 2009 outbreak. Of 30 patients, 12 (80%) of 15 adults and 14 (93%) of 15 children had serious underlying illnesses. Only 12 (57%) of 21 patients who received antiviral therapy did so within 48 hours after symptom onset, but 53% needed escalated care or mechanical ventilation; 8 (27%) of 30 died. Despite national guidelines and standardized infection control procedures, nosocomial transmission remains a problem when influenza is prevalent. Health care workers should be routinely offered influenza vaccine, and vaccination should be prioritized for all patients at high risk. Staff should remain alert to the possibility of influenza in patients with complex clinical problems and be ready to institute antiviral therapy while awaiting diagnosis during influenza outbreaks.
Nosocomial infections; influenza; pandemic (H1N1) 2009; influenza A virus; infection control; hospitalization; viruses; United Kingdom; expedited; research
Regulatory CD4+ T cells have been shown to be important in limiting immune responses, but their role in respiratory viral infections has received little attention. Here we observed that following respiratory syncytial virus (RSV) infection, CD4+ Foxp3+ CD25+ natural regulatory T-cell numbers increased in the bronchoalveolar lavage fluid, lung, mediastinal lymph nodes, and spleen. The depletion of CD25+ natural regulatory T cells prior to RSV infection led to enhanced weight loss with delayed recovery that was surprisingly accompanied by increased numbers of activated natural killer cells in the lung and bronchoalveolar lavage fluid on day 8 postinfection. Increased numbers of neutrophils were also detected within the bronchoalveolar lavage fluid and correlated with elevated levels of myeloperoxidase as well as interleukin-6 (IL-6) and gamma interferon (IFN-γ). CD25+ natural regulatory T-cell depletion also led to enhanced numbers of proinflammatory T cells producing IFN-γ and tumor necrosis factor alpha (TNF-α) in the lung. Despite these increases in inflammatory responses and disease severity, the viral load was unaltered. This work highlights a critical role for natural regulatory T cells in regulating the adaptive and innate immune responses during the later stages of lung viral infections.
Respiratory syncytial virus (RSV) is the main cause of bronchiolitis, the major cause of hospitalization of infants. An ideal RSV vaccine would be effective for neonates, but the immune responses of infants differ markedly from those of adults, often showing a bias toward T-helper 2 (Th2) responses and reduced gamma interferon (IFN-γ) production. We previously developed recombinant RSV vectors expressing IFN-γ and interleukin-4 (IL-4) that allow us to explore the role of these key Th1 and Th2 cytokines during infection. The aim of the current study was to explore whether an immunomodulation of infant responses could enhance protection. The expression of IFN-γ by a recombinant RSV vector (RSV/IFN-γ) attenuated primary viral replication in newborn mice without affecting the development of specific antibody or T-cell responses. Upon challenge, RSV/IFN-γ mice were protected from the exacerbated disease observed for mice primed with wild-type RSV; however, antiviral immunity was not enhanced. Conversely, the expression of IL-4 by recombinant RSV did not affect virus replication in neonates but greatly enhanced Th2 immune responses upon challenge without affecting weight loss. These studies demonstrate that it is possible to manipulate infant immune responses by using cytokine-expressing recombinant viruses and that neonatal deficiency in IFN-γ responses may lead to enhanced disease during secondary infection.
Respiratory syncytial virus (RSV) causes bronchiolitis, the main cause of infantile hospitalization. Immunity against reinfection is poor, and there is great interest in boosting vaccine responses using live vectors expressing host cytokines. We therefore constructed a recombinant RSV expressing murine interleukin 18 (RSV/IL-18), a cytokine capable of inducing strong antiviral immune responses. In vitro RSV/IL-18 replicated at wild-type levels and produced soluble IL-18. In naïve BALB/c mice, RSV/IL-18 infection significantly increased both IL-18 mRNA and protein and attenuated the peak viral load 3-fold. Despite a reduced viral load, RSV/IL-18 infection caused a biphasic weight loss at days 2 and 6 postinfection that was not seen in wild-type infection. Day 2 disease was associated with enhanced pulmonary natural killer (NK) cell numbers and activity and was prevented by NK cell depletion during infection; day 6 disease was correlated with CD8 T-cell recruitment and was enhanced by NK cell depletion. IL-18 expression during priming also enhanced RSV-specific antibody responses and T-cell responses on secondary RSV infection. Therefore, while IL-18 boosted antiviral immunity and reduced the viral load, its coexpression worsened disease. This is the first recombinant RSV with this property, and these are the first studies to demonstrate that NK cells can induce pathology during pulmonary viral infections.
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.
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.
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.
Host defenses, while effecting viral clearance, contribute substantially to inflammation and disease. This double action is a substantial obstacle to the development of safe and effective vaccines against many agents, particularly respiratory syncytial virus (RSV). RSV is a common cold virus and the major cause of infantile bronchiolitis worldwide. The role of αβ T cells in RSV-driven immunopathology is well studied, but little is known about the role of “unconventional” T cells. During primary RSV challenge of BALB/c mice, some Vγ7+ γδ T cells were present; however, immunization with a live vaccinia vector expressing RSV F protein substantially enhanced Vγ4+ γδ T cell influx after RSV infection. Harvested early, these cells produced IFN-γ, TNF, and RANTES after ex vivo stimulation. By contrast, those recruited 5 days after challenge made IL-4, IL-5, and IL-10. Depletion of γδ T cells in vivo reduced lung inflammation and disease severity and slightly increased peak viral replication but did not prevent viral clearance. These studies demonstrate a novel role for γδ T cells in the development of immunopathology and cellular influx into the lungs after immunization and RSV challenge. Though a minor population, γδ T cells have a critical influence on disease and are an attractive interventional target in the alleviation of viral lung disease.
Macrophages are abundant in the lower respiratory tract. They play a central role in the innate response to infection but may also modulate excessive inflammation. Both macrophages and ciliated epithelial cells respond to infection by releasing soluble mediators, leading to the recruitment of innate and adaptive effector cells. To study the role of lung macrophages in acute respiratory viral infection, we depleted them by the inhalation of clodronate liposomes in an established mouse model of respiratory syncytial virus (RSV) disease. Infection caused an immediate local release of inflammatory cytokines and chemokines, peaking on day 1, which was virtually abolished by clodronate liposome treatment. Macrophage depletion inhibited the activation (days 1 to 2) and recruitment (day 4) of natural killer (NK) cells and enhanced peak viral load in the lung (day 4). However, macrophage depletion did not affect the recruitment of activated CD4 or CD8 T cells, weight loss, or virus-induced changes in lung function. Therefore, lung macrophages play a central role in the early responses to viral infection but have remarkably little effect on the adaptive response occurring at the time of peak disease severity.
Respiratory syncytial virus (RSV) is the major cause of infantile bronchiolitis and hospitalization. Severe RSV disease is associated with the development of wheezing in later life. In a mouse model of the delayed effects of RSV, the age at primary infection determines responses to reinfection in adulthood. During primary RSV infection, neonatal BALB/c mice developed only mild disease and recruited CD8 cells that were defective in gamma interferon production. Secondary reinfection of neonatally primed mice caused enhanced inflammation and profuse lung T-cell recruitment. CD4 cell depletion during secondary RSV challenge attenuated disease (measured by weight loss); depletion of CD8 cells also markedly attenuated disease severity but enhanced lung eosinophilia, and depletion of both CD4 and CD8 cells together completely abrogated weight loss. Depletion of CD8 (but not CD4) cells during primary neonatal infection was protective against weight loss during adult challenge. Therefore, T cells, in particular CD8 T cells, play a central role in the outcome of neonatal infection by enhancing disease during secondary challenge. These findings demonstrate a crucial role for T cells in the regulation of immune responses after neonatal infection.
Respiratory syncytial virus (RSV) is an important cause of infant morbidity and mortality worldwide and is increasingly recognized to have a role in the development and exacerbation of chronic lung diseases. There is no effective vaccine, and we reasoned that it might be possible to skew the immune system towards beneficial nonpathogenic responses by selectively priming protective T-cell subsets. We therefore tested recombinant RSV (rRSV) candidates expressing prototypic murine Th1 (gamma interferon [IFN-γ]) or Th2 (interleukin-4 [IL-4]) cytokines, with detailed monitoring of responses to subsequent infections with RSV or (as a control) influenza A virus. Although priming with either recombinant vector reduced viral load during RSV challenge, enhanced weight loss and enhanced pulmonary influx of RSV-specific CD8+ T cells were observed after challenge in mice primed with rRSV/IFN-γ. By contrast, rRSV/IL-4-primed mice were protected against weight loss during secondary challenge but showed airway eosinophilia. When rRSV/IL-4-primed mice were challenged with influenza virus, weight loss was attenuated but was again accompanied by marked airway eosinophilia. Thus, immunization directed toward enhancement of Th1 responses reduces viral load but is not necessarily protective against disease. Counter to expectation, Th2-biased responses were more beneficial but also influenced the pathological effects of heterologous viral challenge.
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.
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.
Respiratory syncytial virus (RSV) is one of the commonest and most troublesome viruses of infancy. It causes most cases of bronchiolitis, which is associated with wheezing in later childhood. In primary infection, the peak of disease typically coincides with the development of specific T- and B-cell responses, which seem, in large part, to be responsible for disease. Animal models clearly show that a range of immune responses can enhance disease severity, particularly after vaccination with formalin-inactivated RSV. Prior immune sensitization leads to exuberant chemokine production, an excessive cellular influx, and an overabundance of cytokines during RSV challenge. Under different circumstances, specific mediators and T-cell subsets and antibody-antigen immune complex deposition are incriminated as major factors in disease. Animal models of immune enhancement permit a deep understanding of the role of specific immune responses in RSV disease, assist in vaccine design, and indicate which immunomodulatory therapy might be beneficial to children with bronchiolitis.
Respiratory syncytial virus (RSV) is a major viral pathogen of infants and the elderly. Significant morbidity is caused by an overexuberant mixed lung cell infiltrate, which is thought to be driven by chemokines. One of the main chemotactic mediators responsible for the movement of eosinophils is CCL11 (eotaxin). Using a mouse model of eosinophilic bronchiolitis induced by RSV, we show here that treatment in vivo with a blocking antibody to CCL11 greatly reduces lung eosinophilia and disease severity. In addition, anti-CCL11 caused a striking inhibition of CD4-T-cell influx and shifted cytokine production away from interleukin-5 without reducing the resistance to viral replication. These results suggest that in addition to influencing eosinophil diapedesis and survival, anti-CCL11 has an action on T cells. These studies strengthen the case for anti-CCL11 treatment of Th2-driven diseases.
Some common childhood infections appear to prevent the development of atopy and asthma. In some Mycobacterium bovis BCG-vaccinated populations, strong delayed-type hypersensitivity responses to mycobacterial antigens are associated with a reduced risk of atopy. Although BCG exposure decreases allergen-induced lung eosinophilia in animal models, little attention has been given to the effect of immunity to BCG on responses against live pathogens. We used the murine Cryptococcus neoformans infection model to investigate whether prior BCG infection can alter such responses. The present study shows that persistent pulmonary BCG infection of C57BL/6 mice induced an increase in gamma interferon, a reduction in interleukin-5, and a decrease in lung eosinophilia during subsequent Cryptococcus infection. This effect was long lasting, depended on the presence of live bacteria, and required persistence of mycobacterial infection in the lung. Reduction of eosinophilia was less prominent after infection with a mutant BCG strain (ΔhspR), which was rapidly cleared from the lungs. These observations have important implications for the development of vaccines designed to prevent Th2-mediated disease and indicate that prior lung BCG vaccination can alter the pattern of subsequent host inflammation.
Infants experiencing severe respiratory syncytial virus (RSV) bronchiolitis have an increased frequency of wheeze and asthma in later childhood. Since most severe RSV infections occur between the 8th and 24th postnatal week, we examined whether age at first infection determines the balance of cytokine production and lung pathology during subsequent rechallenge. Primary RSV infection in newborn mice followed the same viral kinetics as in adults but was associated with reduced and delayed IFN-γ responses. To study rechallenge, mice were infected at 1 day or 1, 4, or 8 weeks of age and reinfected at 12 weeks. Neonatal priming produced more severe weight loss and increased inflammatory cell recruitment (including T helper 2 cells and eosinophils) during reinfection, whereas delayed priming led to enhanced interferon γ production and less severe disease during reinfection. These results show the crucial importance of age at first infection in determining the outcome of reinfection and suggest that the environment of the neonatal lung is a major determinant of cytokine production and disease patterns in later life. Thus, simply delaying RSV infection beyond infancy might reduce subsequent respiratory morbidity in later childhood.
bronchiolitis; asthma; immunity; pneumovirinae; virus
T cells secreting interleukin (IL)-4 and IL-5 (T helper cell type 2 [Th2] cells) play a detrimental role in a variety of diseases, but specific methods of regulating their activity remain elusive. T1/ST2 is a surface ligand of the IL-1 receptor family, expressed on Th2- but not on interferon (IFN)-γ–producing Th1 cells. Prior exposure of BALB/c mice to the attachment (G) or fusion (F) protein of respiratory syncytial virus (RSV) increases illness severity during intranasal RSV challenge, due to Th2-driven lung eosinophilia and exuberant Th1-driven pulmonary infiltration, respectively. We used these polar models of viral illness to study the recruitment of T1/ST2 cells to the lung and to test the effects of anti-T1/ST2 treatment in vivo. T1/ST2 was present on a subset of CD4+ cells from mice with eosinophilic lung disease. Monoclonal anti-T1/ST2 treatment reduced lung inflammation and the severity of illness in mice with Th2 (but not Th1) immunopathology. These results show that inhibition of T1/ST2 has a specific effect on virally induced Th2 responses and suggests that therapy targeted at this receptor might be of value in treating Th2-driven illness.
bronchiolitis, viral; immunity, mucosal; immunity, cellular; pulmonary infection; eosinophil
The effect of infection history is ignored in most animal models of infectious disease. The attachment protein of respiratory syncytial virus (RSV) induces T helper cell type 2–driven pulmonary eosinophilia in mice similar to that seen in the failed infant vaccinations in the 1960s. We show that previous influenza virus infection of mice: (a) protects against weight loss, illness, and lung eosinophilia; (b) attenuates recruitment of inflammatory cells; and (c) reduces cytokine secretion caused by RSV attachment protein without affecting RSV clearance. This protective effect can be transferred via influenza-immune splenocytes to naive mice and is long lived. Previous immunity to lung infection clearly plays an important and underestimated role in subsequent vaccination and infection. The data have important implications for the timing of vaccinations in certain patient groups, and may contribute to variability in disease susceptibility observed in humans.
viral immunology; murine model; eosinophils; major histocompatibility complex tetramers; mucosal immunology