Search

MeCP2 duplication syndrome is a devastating neurodevelopmental disorder caused by duplication of the Xq28 region of the X chromosome, primarily affecting males. Methyl-CpG-binding protein 2 (MeCP2) ubiquitously binds methylated CpG sites in the genome, and regulates gene expression by recruitment of transcriptional mediators. MeCP2 is expressed highly in both neurons and immune cells, thus mutations or over-expression of MeCP2 are associated with neurologic pathology (including seizures, motor dysfunction, and cognitive impairment), and immunologic pathology. Many patients die by the age of 25, and recurrent respiratory infections are a common cause of death. We are studying the pathogenesis of influenza A virus in a mouse model of MeCP2 overexpression, the MeCP2Tg3 mouse, which expresses MeCP2 at ~3 fold normal levels. MeCP2Tg3 mice display significant mortality approximately one week post infection, with T and B cell deficiencies in both number and activation, further characterized by high viral titer and low virus-specific antibody levels in the infected lung. Preliminary evidence suggests that vascular and/or lymphatic impairment may underlie both immunologic defects and the increased mortality observed in MeCP2Tg3 mice during influenza A virus infection. In sum, these findings uncover MeCP2 as an important regulator of host response to respiratory virus infection and may inform treatment of human patients with MeCP2 duplication syndrome.

RSV infection is the most common cause of lower respiratory diseases in infants and young children. It is believed that over-exuberant inflammatory responses are mainly responsible for the disease development. Here, we seek to define host factors that can dampen over-reactive pulmonary inflammation and injury during RSV infection. We have identified that the co-inhibitory receptor PD-1 is up-regulated on pulmonary effector T cells in RSV infected mice and infants. Importantly, acute blockade of PD-1 ligand (PD-L1) function in vivo resulted in the development of excessive pulmonary inflammation and severe host diseases. Using the in vivo and in vitro cytokine staining assays, we found that PD-L1 blockade specifically enhanced the in situ inflammatory activities (i.e. release of inflammatory cytokines) of effector T cells in the lung. Interestingly, we showed that PD-L1 expression is mainly restricted to the monocyte derived dendritic cell (Mo-DC) population in the lung, and that an axis, involving with type I and II interferons (IFN), STAT1 and IRF1, is critical for the induction of PD-L1 expression on Mo-DCs. Collectively, these data suggest that IFN-dependent PD-L1 expression on respiratory DCs is critical to inhibit the pathogenic activities of tissue effector T cells, thereby suppressing the development of excessive pulmonary inflammation and lung injury during RSV infection. Our study also opens the door for novel strategies to treat sever RSV infection.

Influenza A virus (IAV) infection in the respiratory tract triggers a robust immune response, resulting in both virus clearance and lung inflammation and injury. After virus clearance, resolution of lung disease occurs during a distinct recovery period. The costimulatory molecule CD86 can modulate T cell activity during the initiation and effector stages of the host response to IAV infection, but the role of CD86 during recovery is unknown. We found that antibody-mediated CD86 blockade after virus clearance led to a delay in recovery, characterized by increased numbers of lung neutrophils and innate cytokines in the bronchial alveolar lavage fluid (BAL), but no change in conventional IAV-specific T cell responses. However, CD86 blockade led to decreased numbers of FoxP3+ regulatory T cells (Tregs), and adoptive transfer of Tregs into α-CD86 treated mice rescued the effect of the blockade, supporting a role for Tregs in promoting recovery after virus clearance. Direct depletion of Tregs late after infection using DEREG mice mimicked the CD86 blockade phenotype, confirming a role for Tregs during recovery after virus clearance. Finally, we identified neutrophils as a target of Treg suppression, since neutrophil depletion in Treg-depleted DEREG mice reduced excess BAL cytokines. These results show that CD86-dependent Tregs contribute to the resolution of disease after IAV infection, in part by suppressing neutrophil-driven cytokine release into the airways.

The humoral immune response to most respiratory virus infections plays a prominent role in virus clearance and is essential for resistance to reinfection. T follicular helper (TFH) cells are believed to support the development both of a potent primary antibody response and of the germinal center response critical for memory B cell development. Using a model of primary murine influenza A virus (IAV) infection, we demonstrate that a novel APC, the LAPC (Late-activator APC), promotes the TFH response in the draining LNs (dLNs) of the IAV-infected lungs. LAPCs migrate from the infected lungs to the dLN “late,” i.e., 6 d after infection, which is concomitant with TFH differentiation. LAPC migration is CXCR3-CXCL9-dependent, and LAPC triggering of TFH cell development requires ICOS-ICOSL-dependent signaling. In IAV infection, the host immune system control this LAPC-mediated TFH differentiation via IL-21.We have demonstrated that IL-21, a well known TFH differentiation inducing factor, produced by NK T-cells in the dLN acts on lymph node resident T cells to stimulate TNF-α release by the T-cells. TNF-α in turn stimulates dLN resident dendritic cells to secrete CXCL9, a potent chemotactic stimulus for migration of CXCR3 expressing lung resident LAPC into the dLN. It is then the migrant LAPC which acts to modulate TFH differentiation during respiratory viral infection. Our results reveal a novel and previously unsuspected mechanism for regulating anti-viral TFH and humoral responses.

Influenza A virus (IAV) infection can produce severe pulmonary inflammation and injury. The host immune response to infection mediates virus clearance but also contributes to pulmonary inflammation and injury, marking the immune response as a potential therapeutic target after severe infections. Host factors mediating IAV clearance and lung inflammation are well-studied, however, factors controlling recovery from disease following IAV clearance are ill-defined. The costimulatory ligand CD86 mediates interactions between immune cells via its receptors CD28 and CTLA-4. We observed in infected mice that blockade of CD86 signaling in vivo after IAV clearance delays recovery and promotes accumulation of neutrophils in the respiratory tract. Furthermore, CD86 blockade reduces both IL-2 and T regulatory cells (Treg) in the lungs. Depletion of Tregs using DREG mice or Treg depleting antibody after IAV clearance also delays recovery and leads to pulmonary neutrophilia. These data suggest that Tregs may play a crucial role in regulating pulmonary inflammation after IAV infection and are in turn dependent on CD86 ligand engagement. We are continuing to explore the role of CD86 in recovery and resolution of inflammation after IAV infection with the long-term goal of exploring the therapeutic potential of optimizing CD86 signaling to promote Treg function in recovery from infection.

Interleukin (IL)-5 is a prototypical type II cytokine that is essential for eosinophil maturation and egress out of the bone marrow. We observed that eosinophil numbers progressively increase in the lungs during the course of experimental influenza A virus (IAV) infection in mice as a result of high level production of IL-5 by natural helper cells recruited to the infected lungs. Mice genetically deficient in IL-5 (IL-5 KO) demonstrate increased lung neutrophil numbers and decreased viral titers during the acute phase of IAV infection. However, this phenotype is not recapitulated in mice deficient in eosinophils (PHIL mice) suggesting an eosinophil independent function of IL-5 during infection. Unexpectedly, we found that like eosinophils, >98% of neutrophils express the IL-5 receptor alpha subunit (CD125) in the lung throughout infection. Unlike eosinophils, where ligation of the IL-5R by IL-5 induces phosphorylation of STAT1 and STAT5, engagement of the IL-5R on activated neutrophils results in suppressed pSTAT5 and pSTAT1 signalling as well as decreased expression of the activation marker CD11b. Furthermore, neutrophils treated with IL-5 undergo accelerated cell death in vitro suggesting a potential mechanism for the enhanced neutrophil numbers seen in the IAV infected IL-5 KO. Overall these data suggest that IL-5 may serve a regulatory role during the acute phase of IAV infection by controlling the number (viability) and activation state of recruited neutrophils.

An effective host response to a microbial pathogen requires both the generation of effector T cells as well as formation of durable T cell memory. Dendritic cells (DCs) play a central role in initiating the adaptive T cell response to microbial infection, and multiple distinct DC subsets have the potential to take up and present microbial antigen. However, the contribution of different DC subsets to the generation of effector and memory CD8+ T cells in vivo following infection and the DC dependent mechanisms to account for this dichotomy remain incompletely understood. Here we show that the two major migratory respiratory DC (RDC) subsets responding in the lung draining lymph nodes to influenza virus infection, CD103+ and CD11bhi RDC, activate naïve virus-specific CD8+ T cells along distinctly different differentiation pathways. CD103+ RDC in vivo stimulate the generation of activated CD8+ T cells with features characteristic of CD8+ T effectors that is activated T cells which efficiently leave the lymph nodes and home to the infected respiratory tract. In contrast, stimulation of naïve virus-specific CD8+ T cells by CD11bhi RDC give rise to CD8+ T effectors which are preferentially retained within the draining lymph nodes during the acute phase of infection, and which exhibits properties of central memory CD8+ cells. DC-dependent cellular and molecular basis for refining this dichotomy for in vivo CD8+ T cell differentiation are further delineated.

Influenza A virus (IAV) is a major human pathogen. Patients with lethal primary influenza pneumonia exhibit severe diffuse lung inflammation, respiratory tract injury, and diffuse alveolar damage. While IAV is cytolytic, the host immune response has recently been appreciated to also be a significant cause of lung injury during IAV infections. To explore the role of alveolar macrophages (AMs) in virus clearance and lung injury/repair during IAV infection, we developed a novel mutant mouse strain with a selective deficiency in the AM compartment. We found that AM deficient mice die following a sub-lethal (in control mice) IAV infection in spite of a normal immune response and virus clearance. However, these mice undergo a rapid decline in O2 saturation at the onset of the adaptive immune response (5-6 dpi) and also demonstrate an increased susceptibility of alveolar type I epithelial cells to IAV infection. Destruction of this critical cell type necessary for oxygen exchange by virus or the adaptive response likely accounts for their loss of lung function and death. Thus AMs may have a critical function in controlling the susceptibility of alveolar epithelial cells to IAV infection and therefore the severity of infection.

Acute viral infections induce robust adaptive immune responses resulting in virus clearance. Recent evidence suggests that there may be depots of viral antigen which persist in draining lymph nodes following virus clearance and could therefore affect the adaptive immune response and memory formation. The nature of these residual antigen depots, the mechanism of antigen persistence and impact of the persistent antigen on memory T cells remains ill-defined. Employing a murine model of influenza virus infection of the respiratory tract, we have identified respiratory dendritic cells (RDC) as an essential cell responsible for both sampling and presenting residual viral antigen. RDC in the previously infected lung capture residual viral antigen deposited in an irradiation resistant cell type(s) and transport the viral antigen to the local lymph nodes draining the site of infection where the migrant RDC present the antigen to T cells. Furthermore, we demonstrate that there is preferential localization of memory T cells to the DLN following virus clearance as a consequence of presentation of residual viral antigen by the migrant RDC to T cells in the DLN. This enriched population of antigen-specific T cells maintained in the antigen draining LN would likely provide a more rapid and robust recall T cell response to a re-infection.

CD8+ cytolytic T lymphocytes (CTLs) play a crucial role in the resolution of respiratory viral infections through their ability to eliminate infected cells and to release potent anti-viral cytokines. In the influenza viral model, CD8+ CTL effector activity is believed to be focused on respiratory epithelial cells, since these cells are the principal cell types supporting influenza replication. Utilizing the prototypical T cell anti-viral cytokine, IFNγ, as a measure of CD8+ CTL effector activity, we evaluated the tempo of the CD8+ CTL response to influenza infection in vivo in two compartments, the airways overlying the respiratory epithelium and the infected lung interstitium. Surprisingly, CD8+ CTL effector activity was localized not to the respiratory epithelium but within the pulmonary interstitium. We were able to establish, utilizing bone marrow chimeric mice, that infiltrating inflammatory cells of hematopoietic origin were the primary stimulus of CD8+ CTL effector activity. Additionally, we further demonstrated that lung resident CD11c+ cells, likely macrophages and/or dendritic cells, were triggering CD8+ CTL anti-viral activity. These observations suggest that inflammatory mononuclear cells are serving as the primary antigen presenting cells for effector CD8+ CTLs responding within the lungs to infection. CD8+ CTL recognition of infected respiratory epithelial cells appears to serve only a secondary role in orchestrating the pro-inflammatory T cell response.

Respiratory syncytial virus (RSV) infection is the leading viral cause of severe lower respiratory tract illness in young infants. Clinical studies have documented that genetic polymorphism of a regulatory cytokine IL-10 is associated with the development of severe bronchiotis in RSV infected infants. However the tempo, source and function of IL-10 during the RSV infection remain unclear. Here, we have examined the role of IL-10 in a murine model of primary RSV infection. We found that high levels of IL-10 are produced in the respiratory tract at the onset of adaptive immune responses. The main cellular sources of the IL-10 are infiltrating anti-viral effector CD4+ and CD8+ T cells (Te). We showed that the function of the Te-derived IL-10 in vivo is mainly to limit the pro-inflammatory activity of the anti-viral Te themselves and thus to control the exaggerated pulmonary inflammation induced by the infiltrating Te. Furthermore, we demonstrated that the inhibition of the exaggerated inflammation by IL-10 is essential in maintaining normal pulmonary function of the host. Our data not only discovered a novel function of IL-10 (i.e., autocrine inhibition of the inflammatory activity of Te) during infection but provided potential explanation and cellular mechanism for the early clinical studies demonstrating a link between IL-10 gene polymorphism and the development of pulmonary diseases during RSV infection.

Influenza virus infection of the respiratory tract triggers a rapid and vigorous innate immune response. Monocyte/macrophages make up a large fraction of this inflammatory infiltrate and are the major contributors to lung inflammation associated with infection. The monocyte population can be defined as CD11b+Ly6G-Dx5-SiglecF-, and subsets can be further discriminated based on Ly6C expression levels. Here we show rapid accumulation of Ly6Chi monocytes in the infected lungs during the inductive phase of the host immune response, resulting in the predominant if not exclusive accumulation of the Ly6Chi monocyte population at the peak of the inflammatory response. The Ly6Chi monocytes are potent producers of TNF alpha and their presence during early infection strongly correlates with overall lung inflammation. Ly6Chi monocytes slowly decline during the recovery phase following virus clearance, resulting in an accumulation of a Ly6Clo monocyte subset. The presence of this Ly6Clo subset, and the subsequent down-regulation of TNF alpha production in the Ly6Chi subset, strongly correlates with recovery (e.g. weight gain and reduced inflammation) in this mouse model of influenza infection.

Influenza A virus is a major cause of infection of the respiratory tract worldwide. Approximately 10–20% of U.S. population is estimated to be infected by this virus each year. Respiratory tract dendritic cells (RDC) are believed to play a major role as antigen presenting cells in initiating adaptive immune response to influenza virus infection. We have examined the effect of influenza virus infection of purified RDC in vitro at different MOI (Multiplicity of Infection) on virus protein expression, co-stimulatory molecules expression, and cytokines production. We found that there is not only an MOI dependent increase in the percentage of infected RDC, but also the infection of RDC at high MOI results in higher level and accelerated viral protein synthesis at early time post infection. Production of tested cytokines and chemokines by RDC increases with increasing of MOI except for IL-12 p40. Furthermore, we found that at high MOI total RDCs can be divided to several populations based on CD40 and influenza nucleoprotein (NP) expression: CD40high NPlow, CD40low NPhigh, and CD40negNPnegDCs. In conclusion, our results suggest that there is a diverse response of RDC to influenza virus at different MOI as reflected by their susceptibility to infection and cytokine production. These results may provide insight into how RDC modulate host response in the lung to influenza virus infection. (Support: USPHS grant, NIH AI-15608, HL-33391,HL-71875)