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1.  CxCL10/CxCR3-mediated Responses Promote Immunity to Respiratory Syncytial Virus Infection by Augmenting Dendritic Cell and CD8+ T Cell Efficacy 
European journal of immunology  2008;38(8):2168-2179.
The induction of inflammatory cytokines during respiratory viral infections contributes to both disease pathogenesis and resolution. The present studies investigated the role of the chemokine CxCL10 and its specific receptor, CxCR3, in the host response to pulmonary respiratory syncytial virus (RSV) infection. Antibody-mediated neutralization of CxCL10 resulted in a significant increase in disease pathogenesis, including airway hyperresponsiveness (AHR), mucus gene expression, and impaired viral clearance. When the pulmonary cytokine levels were examined, only type I IFN and IL-12p70 were significantly reduced. These latter observations were reflected in reduced dendritic cell (DC) numbers and DC maturation in the lungs of RSV-infected mice treated with anti-CxCL10. Neutralization of the only known receptor for CxCL10, CxCR3, resulted in similar increases in pathogenic responses. When bone marrow-derived DC (BMDC) were incubated with CxCL10 and RSV, an upregulation of type I IFN was observed. In addition, T lymphocytes were also examined and a significant decrease in the number of RSV M2 peptide-specific CD8+ T cells was identified. These findings highlight a previously unappreciated role for the CXCL10:CXCR3 signaling axis in RSV-infected animals by recruiting virus-specific T cells into the lung and promoting viral clearance.
PMCID: PMC2743117  PMID: 18624292
Rodent; Lung; Dendritic Cells; Chemokines; Cytokines
2.  Autophagy-mediated DC activation is essential for innate cytokine production and APC function with Respiratory Syncytial Virus responses 
The regulation of innate immune responses during viral infection is a crucial step to promote anti-viralreactions. Recent studies have drawn attention to a strong relationship of pathogen associated molecular patterns (PAMP) recognition with autophagy for activation of APC function. Our initial observations indicated that autophagosomes formed in response to RSV infection of DC. To further investigate whether RSV-induced DC activation and innate cytokine production was associated with autophagy, we utilized several methods to block autophagosome formation. Using 3-MA,siRNA inhibition of LC3,or Beclin +/- mouse derived DC,studies establisheda relationship between RSV-induced autophagy and enhanced type I IFN, TNF, IL-6, and IL-12p40expression. Moreover, autophagosome formation induced by starvation also promoted innate cytokine expression in DC. The induction of starvation-induced autophagy in combination with RSV infection synergistically enhanced DC cytokine expressionthat was blocked by an autophagy inhibitor. The latter synergistic responses were differentially altered in DC from MyD88-/- and TRIF-/-mice supporting the concept of autophagy-mediated TLR signaling. In addition, blockade of autophagy in RSV-infected DC inhibited the maturation of DCs as assessed by MHC Class II and co-stimulatory molecule expression. Subsequently, we demonstrated that inhibition of autophagy in DCsused to stimulateprimary ovalbumin-induced and secondary RSV-infected responses significantly attenuatedcytokine production by CD4+ T cells. Thus, these studies have outlined that autophagy in DC afterRSV infection isa crucial mechanism for driving innate cytokine productionleading to alteredacquired immune responses.
PMCID: PMC3186849  PMID: 21911604
3.  Respiratory Virus-induced TLR7 activation controls IL-17 associated Increase in mucus via IL-23 regulation 
The response to respiratory syncytial virus (RSV), negative strand ssRNA virus, depends upon the ability to recognize specific pathogen associated targets. In the present study the role of TLR7 that recognizes ssRNA was examined. Using TLR7−/− mice we found that the response to RSV infection in the lung was more pathogenic as assessed by significant increases in inflammation and mucus hyper-secretion. While there appeared to be no effect of TLR7 deficiency on Type I IFN, the pathology was associated with an alteration in T cell responses with increases in mucogenic cytokines, IL-4, IL-13 and IL-17. Examination of DC from TLR7−/− animals indicated a preferential activation of IL-23 (a Th17 associated cytokine) and a decrease in IL-12 production. Neutralization of IL-17 in the TLR7−/− mice resulted in a significant decrease in the mucogenic response in the lungs of the RSV-infected mice. Thus, without TLR7-mediated responses an altered immune environment ensued with a significant effect on airway epithelial cell remodeling and goblet cell hyper/metaplasia leading to mucus overproduction.
PMCID: PMC3006454  PMID: 20624950
4.  A Novel Inactivated Intranasal Respiratory Syncytial Virus Vaccine Promotes Viral Clearance without Th2 Associated Vaccine-Enhanced Disease 
PLoS ONE  2011;6(7):e21823.
Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960's led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations.
Principal Findings
In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines.
These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.
PMCID: PMC3137595  PMID: 21789184
5.  CCR6 deficiency enhances immunity to respiratory syncytial virus by impairing recruitment of T helper type II-skewing dendritic cells 
European journal of immunology  2010;40(4):1042-1052.
Chemokines are important mediators of the immune response to pathogens, but can also promote chronic inflammatory states. CCR6 is a chemokine receptor found on dendritic cells and T cells, suggesting its role in both innate and adaptive immunity. We investigated the role of CCR6 in a pulmonary viral infection caused by respiratory syncytial virus (RSV), a ubiquitous virus that can cause severe pulmonary complications. We found that CCR6−/− mice had reduced pathophysiology and lower numbers of activated T cells in the lungs post-RSV challenge. Analysis of RSV-specific cytokine responses showed that CCR6−/− mice skewed preferentially toward a Th1 effector response. Correspondingly, viral clearance was rapid and early in CCR6−/− mice, suggesting an efficient immune response. Early timepoint analysis revealed that CCR6−/− mice had significantly fewer conventional DCs (cDCs) recruited into the lung, but the same number of plasmacytoid DCs. A pathogenic phenotype could be reconstituted in CCR6−/− mice by supplying cDCs into the airway, indicating that mere number of cDCs dictates the adverse response. Our data suggest that CCR6 deficiency provides an environment whereby the balance of innate immune cells mediates the efficient antiviral response to RSV.
PMCID: PMC2952176  PMID: 20101616
dendritic cells; viral; chemokines; inflammation; lung
6.  Delta-Like 4 Differentially Regulates Murine CD4+ T Cell Expansion via BMI1 
PLoS ONE  2010;5(8):e12172.
Studies have shown that Notch is essential for the maintenance of a T cell Th2 phenotype in vivo. It has also been shown that Notch ligands have diverse functions during T cell activation. We chose to investigate the role of Notch ligands during the Th2 response.
Principal Findings
We studied the relationship of two Notch ligands, delta-like 4 and jagged-1, to T cell proliferation in C57 Bl/6 mice. Our findings indicate that jagged-1 does not affect the rate of T cell proliferation in any subset examined. However, delta-like 4 causes an increase in the expansion of Th2 memory cells and a decrease in effector cell proliferation. Our in vivo studies indicate that the Notch system is dynamically regulated, and that blocking one Notch ligand increases the effective concentration of other Notch ligands, thus altering the response. Examination of genes related to the Notch pathway revealed that the Notch receptors were increased in memory T cells. Expression of BMI1, a gene involved in T cell proliferation, was also higher in memory T cells. Further experiments demonstrated that Notch directly regulates the expression of the BMI1 gene in T cells and may govern T cell proliferation through this pathway.
From these experiments we can make several novel conclusions about the role of Notch ligands in T cell biology. The first is that delta-like 4 suppresses effector cell proliferation and enhances Th2 memory cell proliferation. The second is that blocking one Notch ligand in vivo effectively increases the concentration of other Notch ligands, which can then alter the response.
PMCID: PMC2923143  PMID: 20808960
7.  B Cell Antigen Presentation Promotes Th2 Responses and Immunopathology during Chronic Allergic Lung Disease 
PLoS ONE  2008;3(9):e3129.
The role of B cells in allergic asthma remains undefined. One mechanism by which B cells clearly contribute to allergic disease is via the production of specific immunoglobulin, and especially IgE. Cognate interactions with specific T cells result in T cell help for B cells, resulting in differentiation and immunoglobulin secretion. Proximal to (and required for) T cell-dependent immunoglobulin production, however, is antigen presentation by B cells. While interaction with T cells clearly has implications for B cell function and differentiation, this study investigated the role that B cells have in shaping the T cell response during chronic allergic lung disease.
Methodology/Principal Findings
In these studies, we used a clinically relevant mouse model of chronic allergic lung disease to study the role of B cells and B cell antigen presentation in this disease. In these studies we present several novel findings: 1) Lung B cells from chronically allergen challenged mice up-regulated MHC II and costimulatory molecules CD40, CD80 and CD86. 2) Using in vitro studies, B cells from the lungs of allergen challenged mice could present antigen to T cells, as assessed by T cell proliferation and the preferential production of Th2 cytokines. 3) Following chronic allergen challenge, the levels of Th2 cytokines IL-4 and IL-5 in the lungs and airways were significantly attenuated in B cell −/− mice, relative to controls. 4) B cell driven Th2 responses and mucus hyper secretion in the lungs were dependent upon MHC II expression by B cells.
Collectively, these results provide evidence for antigen presentation as a novel mechanism by which B cells contribute to chronic allergic disease. These findings give new insight into the mechanisms by which B cells promote asthma and other chronic diseases.
PMCID: PMC2518863  PMID: 18769622
8.  The Balance between Plasmacytoid DC versus Conventional DC Determines Pulmonary Immunity to Virus Infections 
PLoS ONE  2008;3(3):e1720.
Respiratory syncytial virus (RSV) infects nearly all infants by age 2 and is a leading cause of bronchiolitis. RSV may employ several mechanisms to induce immune dysregulation, including dendritic cell (DC) modulation during the immune response to RSV.
Methods and Findings
Expansion of cDC and pDC by Flt3L treatment promoted an anti-viral response with reduced pathophysiology characterized by decreased airway hyperreactivity, reduced Th2 cytokines, increased Th1 cytokines, and a reduction in airway inflammation and mucus overexpression. These protective aspects of DC expansion could be completely reversed by depleting pDCs during the RSV infection. Expansion of DCs by Flt3L treatment enhanced in CD8+ T cell responses, which was reversed by depletion of pDC.
These results indicate that a balance between cDC and pDC in the lung and its lymph nodes is crucial for the outcome of a pulmonary infection. Increased pDC numbers induced by Flt3L treatment have a protective impact on the nature of the overall immune environment.
PMCID: PMC2249704  PMID: 18320041
9.  Diversity of the T-Cell Response to Pulmonary Cryptococcus neoformans Infection  
Infection and Immunity  2006;74(8):4538-4548.
Cell-mediated immunity plays an important role in immunity to the pathogenic fungus Cryptococcus neoformans. However, the antigen specificity of the T-cell response to C. neoformans remains largely unknown. In this study, we used two approaches to determine the antigen specificity of the T-cell response to C. neoformans. We report here that a diverse T-cell receptor (TCR) Vβ repertoire was maintained throughout the primary response to pulmonary C. neoformans infection in immunocompetent mice. CD4+ T-cell deficiency resulted in relative expansion of all CD8+ T-cell subsets. During a secondary immune response, preferential usage of a TCR Vβ subset in CD4+ T cells occurred in single individuals, but the preferences were “private” and not shared between individuals. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of immunized mice proliferated in response to a variety of C. neoformans antigens, including heat-killed whole C. neoformans, culture filtrate antigen, C. neoformans lysate, and purified cryptococcal mannoprotein. CD4+ and CD8+ T cells from the secondary lymphoid tissues of mice undergoing a primary response to C. neoformans proliferated in response to C. neoformans lysate. In response to stimulation with C. neoformans lysate, lung CD4+ and CD8+ T cells produced the effector cytokines tumor necrosis factor alpha and gamma interferon. These results demonstrate that a diverse T-cell response is generated in response to pulmonary C. neoformans infection.
PMCID: PMC1539621  PMID: 16861640
10.  Macrophage Inflammatory Protein 1α/CCL3 Is Required for Clearance of an Acute Klebsiella pneumoniae Pulmonary Infection 
Infection and Immunity  2001;69(10):6364-6369.
The objective of these studies was to determine the role of macrophage inflammatory protein 1α/CCL3 in pulmonary host defense during Klebsiella pneumoniae infection. Following intratracheal inoculation, 7-day survival of CCL3−/− mice was less than 10%, compared to 60% for CCL3+/+ mice. Survival of CCR5−/− mice was equivalent to that of controls, indicating that the enhanced susceptibility of CCL3−/− mice to K. pneumoniae is mediated via another CCL3 receptor, presumably CCR1. At day 3, CFU burden in the lungs of CCL3−/− mice was 800-fold higher than in CCL3+/+ mice, demonstrating that CCL3 is critical for control of bacterial growth in the lung. Surprisingly, CCL3−/− mice had no differences in the recruitment of monocytes/macrophages and even showed enhanced neutrophil recruitment at days 1, 2, and 3 postinfection, compared to CCL3+/+ mice. Therefore, the defect in clearance was not due to insufficient recruitment of leukocytes. No significant differences in cytokine levels of monocyte chemoattractant protein 1 (MCP-1), interleukin 12, gamma interferon, or tumor necrosis factor alpha in lung lavages were found between CCL3+/+ and CCL3−/− mice. CCL3−/− alveolar macrophages were found to have significantly lower phagocytic activity toward K. pneumoniae than CCL3+/+ alveolar macrophages. These findings demonstrate that CCL3 production is critical for activation of alveolar macrophages to control the pulmonary growth of the gram-negative bacterium K. pneumoniae.
PMCID: PMC98771  PMID: 11553580

Results 1-10 (10)