Regulatory T Cells Prevent Th2 Immune Responses and Pulmonary Eosinophilia during Respiratory Syncytial Virus Infection in Mice

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 Foxp3(DTR) 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

Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection

The inflammatory response to lung infections must be tightly regulated, enabling pathogen elimination while maintaining crucial gas exchange. Using recently described “depletion of regulatory T cell” (DEREG) mice, we found that selective depletion of regulatory T cells (Tregs) during acute respiratory syncytial virus (RSV) infection enhanced viral clearance but increased weight loss, local cytokine and chemokine release, and T-cell activation and cellular influx into the lungs. Conversely, inflammation was decreased when Treg numbers and activity were boosted using interleukin-2 immune complexes. Unexpectedly, lung (but not draining lymph node) Tregs from RSV-infected mice expressed granzyme B (GzmB), and bone marrow chimeric mice with selective loss of GzmB in the Treg compartment displayed markedly enhanced cellular infiltration into the lung after infection. A crucial role for GzmB-expressing Tregs has not hitherto been described in the lung or during acute infections, but may explain the inability of children with perforin/GzmB defects to regulate immune responses to infection. The effects of RSV infection in mice with defective immune regulation closely parallel the observed effects of RSV in children with bronchiolitis, suggesting that the pathogenesis of bronchiolitis may involve an inability to regulate virus-induced inflammation

IL-10 Regulates Viral Lung Immunopathology during Acute Respiratory Syncytial Virus Infection in Mice

Interleukin (IL-) 10 is a pleiotropic cytokine with broad immunosuppressive functions, particularly at mucosal sites such as the intestine and lung. Here we demonstrate that infection of BALB/c mice with respiratory syncytial virus (RSV) induced IL-10 production by CD4(+) and CD8(+) T cells in the airways at later time points (e.g. day 8); a proportion of these cells also co-produced IFN-γ. Furthermore, RSV infection of IL-10(−/−) mice resulted in more severe disease with enhanced weight loss, delayed recovery and greater cell infiltration of the respiratory tract without affecting viral load. In addition, IL-10(−/−) mice had a pronounced airway neutrophilia and heightened levels of pro-inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid. Notably, the proportion of lung T cells producing IFN-γ was enhanced, suggesting that IL-10 may act in an autocrine manner to dampen effector T cell responses. Similar findings were made in mice treated with anti-IL-10R antibody and infected with RSV. Therefore, IL-10 inhibits disease and inflammation in mice infected with RSV, especially during recovery from infection

Alveolar macrophage-derived type I interferons orchestrate innate immunity to RSV through recruitment of antiviral monocytes

Type I interferons (IFNs) are important for host defense from viral infections, acting to restrict viral production in infected cells and to promote antiviral immune responses. However, the type I IFN system has also been associated with severe lung inflammatory disease in response to respiratory syncytial virus (RSV). Which cells produce type I IFNs upon RSV infection and how this directs immune responses to the virus, and potentially results in pathological inflammation, is unclear. Here, we show that alveolar macrophages (AMs) are the major source of type I IFNs upon RSV infection in mice. AMs detect RSV via mitochondrial antiviral signaling protein (MAVS)–coupled retinoic acid–inducible gene 1 (RIG-I)–like receptors (RLRs), and loss of MAVS greatly compromises innate immune restriction of RSV. This is largely attributable to loss of type I IFN–dependent induction of monocyte chemoattractants and subsequent reduced recruitment of inflammatory monocytes (infMo) to the lungs. Notably, the latter have potent antiviral activity and are essential to control infection and lessen disease severity. Thus, infMo recruitment constitutes an important and hitherto underappreciated, cell-extrinsic mechanism of type I IFN–mediated antiviral activity. Dysregulation of this system of host antiviral defense may underlie the development of RSV-induced severe lung inflammation

Defective immunoregulation in RSV vaccine-augmented viral lung disease restored by selective chemoattraction of regulatory T cells

Human trials of formaldehyde-inactivated respiratory syncytial virus (FI-RSV) vaccine in 1966–1967 caused disastrous worsening of disease and death in infants during subsequent natural respiratory syncytial virus (RSV) infection. The reasons behind vaccine-induced augmentation are only partially understood, and fear of augmentation continues to hold back vaccine development. We now show that mice vaccinated with FI-RSV show enhanced local recruitment of conventional CD4(+) T cells accompanied by a profound loss of regulatory T cells (Tregs) in the airways. This loss of Tregs was so complete that additional depletion of Tregs (in transgenic depletion of regulatory T-cell mice) produced no additional disease enhancement. Transfer of conventional CD4(+) T cells from FI-RSV–vaccinated mice into naive RSV-infected recipients also caused a reduction in airway Treg responses; boosting Tregs with IL-2 immune complexes failed to restore normal levels of Tregs or to ameliorate disease. However, delivery of chemokine ligands (CCL) 17/22 via the airway selectively recruited airway Tregs and attenuated vaccine-augmented disease, reducing weight loss and inhibiting local recruitment of pathogenic CD4(+) T cells. These findings reveal an unexpected mechanism of vaccine-induced disease augmentation and indicate that selective chemoattraction of Tregs into diseased sites may offer a novel approach to the modulation of tissue-specific inflammation

Live Attenuated B. pertussis BPZE1 Rescues the Immune Functions of Respiratory Syncytial Virus Infected Human Dendritic Cells by Promoting Th1/Th17 Responses

Respiratory Syncytial virus (RSV) is the leading cause of acute lower respiratory tract viral infection in young children and a major cause of winter hospitalization. Bordetella pertussis is a common cause of bacterial lung disease, affecting a similar age group. Although vaccines are available for B. pertussis infection, disease rates have recently increased in many countries. We have therefore developed a novel live attenuated B. pertussis strain (BPZE1), which has recently undergone a successful clinical phase I trial. In mice, BPZE1 provides protection against disease caused by respiratory viral challenge. Here, we analyze the effect of BPZE1 on antiviral T cell responses induced by human monocyte-derived dendritic cells (MDDC). We found that BPZE1 influences antiviral immune responses at several levels, enhancing MDDC maturation, IL-12p70 production, and shifting T cell cytokine profile towards a Th1/Th17 pattern. These data were supported by the intracellular signaling analysis. RSV infection of MDDC caused MyD88-independent STAT1 phosphorylation, whereas BPZE1 activated MyD88-dependent signaling pathways; co-infection caused both pathways to be activated. These findings suggest that BPZE1 given during infancy might improve the course and outcome of viral lung disease in addition to providing specific protection against B. pertussis infection

Blocking IL-10R signalling increases cellular influx into the lungs and delays recovery during RSV infection.

<p>BALB/c mice were infected with 10⁶ PFU RSV i.n. (day 0). Where indicated, mice were injected with anti-IL-10R antibody on day -1 (i.p.), 3 (i.p. and i.n) and 6 (i.p.) post RSV infection. Control groups were injected with rat IgG. (A) Illness was monitored daily by changes in weight for 8 days after RSV infection; the percentage of original weight is shown. (B) Viral titer was measured in the lung on day 4 post infection by quantifying RSV L gene copies by qPCR. (C) Total numbers of cells in the lung and BAL were enumerated on day 4 and 8 from naïve or RSV infected mice. (D) Total numbers of neutrophils in the BAL were quantified using differential cell counting of H&E stained cytospins slides on day 4 and 8 post infection. (E) Total numbers of NK cells (CD3⁻ NKp46⁺) and CD3-gated, CD4⁺Foxp3⁻ and CD8⁺ T cells in the lung were quantified using flow cytometry on day 4 and 8 post RSV infection. Error bars indicate the SEM. The data are representative of two independent experiments with n = 4–5 mice per group.</p