The predominant immune response to allergens in atopic asthma is mediated by T cells of the helper subtype 2 (Th2). In the past few years, we and others have made significant contributions to our current understanding of Th2 differentiation. Despite these advances, the mechanisms that prevent the generation of Th2 responses in the respiratory tract are less well understood. Using a model of tolerance induced by repeated aerosol exposures to Ag, we have shown: 1) inhibition of airway inflammation in tolerized mice upon Ag challenge; 2) inhibition of GATA-3 and NFAT expression in CD4+ T cells in recall responses to Ag; 3) ability of splenic cells from tolerized mice to inhibit immune responses in recipient mice. In vitro studies, the immunosuppressive cytokines TGF-beta, which has been implicated in oral tolerance, inhibited nuclear translocation of NFAT proteins and GATA-3 expression. Collectively, these observations lead us to hypothesize that: a) self-regulatory active mechanisms of suppression can be elicited upon repeated exposure to Ag in the respiratory tract; b) active suppression involves induction of negative signaling pathways that in turn inhibit expression/activation of critical transcription factors such as NFAT and GATA-3. To address this hypothesis we will:
Aim 1. Characterize the features of immunosuppression in tolerized mice and identify the cell type(s) that mediate immunosuppression. a) The dose of Ag and the length of exposure required to induce tolerance will be investigated. b) The fate of Th2 cells derived from TCR Tg mice will be followed upon adoptive transfer in tolerized mice. c) Cells from spleen and lung-draining lymph nodes from tolerized mice will be fractionated into CD4+, CD8+, and gamma-delta cells to identify the cell type that can confer immunosuppression in vivo.
Aim II. Characterize the signaling pathways that are induced in tolerized/immunosuppressed CD4+ T cells. a) CD4+ T cells from spleen or lung-draining lymph nodes of tolerized mice will be restimulated and analyzed for activation/induction of transcription factors including Stat6, GATA-3, NF-AT, NF-kB, and AP-1. b) CD4+ T cells from naive mice will be treated with culture supernatants of cells from tolerized mice and analyzed as in a). c) Cells in a) and b) will be measured for (Ca2+)i levels and calcineurin activity and GATA-3 mRNA expression. d) The activation of MAPK and Stat 5 will be investigated given their critical role in TCR-directed gene expression and responsiveness to Ag.
Aim III. Investigation of tolerance in mice with defined genetic alterations. a) IL-10-/- mice and mice treated with anti-TGF-beta; b) mice expressing a dominant-negative mutant of GATA-3; and c) mice treated with CTLA4-Ig fusion protein will be analyzed in the tolerance model; d) mice over expressing NFAT or GATA-3 in an inducible and T cell-specific fashion will be tested for their ability to overcome tolerance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI048927-06
Application #
6838198
Study Section
Special Emphasis Panel (ZRG1-RAP (01))
Program Officer
Minnicozzi, Michael
Project Start
2001-01-01
Project End
2005-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
6
Fiscal Year
2005
Total Cost
$352,701
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Oriss, Timothy B; Raundhal, Mahesh; Morse, Christina et al. (2017) IRF5 distinguishes severe asthma in humans and drives Th1 phenotype and airway hyperreactivity in mice. JCI Insight 2:
Das, Sudipta; Raundhal, Mahesh; Chen, Jie et al. (2017) Respiratory syncytial virus infection of newborn CX3CR1-deficent mice induces a pathogenic pulmonary innate immune response. JCI Insight 2:
Gauthier, Marc; Chakraborty, Krishnendu; Oriss, Timothy B et al. (2017) Severe asthma in humans and mouse model suggests a CXCL10 signature underlies corticosteroid-resistant Th1 bias. JCI Insight 2:
Chakraborty, Krishnendu; Raundhal, Mahesh; Chen, Bill B et al. (2017) The mito-DAMP cardiolipin blocks IL-10 production causing persistent inflammation during bacterial pneumonia. Nat Commun 8:13944
Ray, Anuradha; Raundhal, Mahesh; Oriss, Timothy B et al. (2016) Current concepts of severe asthma. J Clin Invest 126:2394-403
Khare, Anupriya; Raundhal, Mahesh; Chakraborty, Krishnendu et al. (2016) Mitochondrial H2O2 in Lung Antigen-Presenting Cells Blocks NF-?B Activation to Prevent Unwarranted Immune Activation. Cell Rep 15:1700-14
Gauthier, Marc; Ray, Anuradha; Wenzel, Sally E (2015) Evolving Concepts of Asthma. Am J Respir Crit Care Med 192:660-8
Khare, Anupriya; Chakraborty, Krishnendu; Raundhal, Mahesh et al. (2015) Cutting Edge: Dual Function of PPAR? in CD11c+ Cells Ensures Immune Tolerance in the Airways. J Immunol 195:431-5
Ray, Anuradha; Oriss, Timothy B; Wenzel, Sally E (2015) Emerging molecular phenotypes of asthma. Am J Physiol Lung Cell Mol Physiol 308:L130-40
Raundhal, Mahesh; Morse, Christina; Khare, Anupriya et al. (2015) High IFN-? and low SLPI mark severe asthma in mice and humans. J Clin Invest 125:3037-50

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