Project Summary: Legionellosis (Legionnaires’ disease) is a form of atypical pneumonia with a steep rising incidence in the United States. The mortality rates of patients stagnate around 3-30% despite treatment. Legionellosis is caused by facultative, intracellular bacteria of the Legionellaceae family, which reside in natural and engineered aquatic habitats. Inhalation of contaminated aerosols can lead to infection, evasion of pathogen eradication and continuous replication in Legionella containing vacuoles inside alveolar macrophages (AMs). The ensuing inflammation promotes an infiltration of myeloid cells and lymphocytes into lungs. These immune cells communicate with each other via cytokines. Interleukin-27 (IL-27) is a heterodimeric cytokine formed by non-covalent interactions of the subunits p28 and EBI3. IL-27 is induced by pattern recognition receptor (PRR) activation in mononuclear phagocytes (AMs, monocytes, dendritic cells). IL-27 ligation with its unique receptor chain, IL-27RA, on lymphocytes initiates STAT1/STAT3 phosphorylation. IL-27RA signaling initiates pleiotropic programs, which can first intensify acute inflammation and later limit prolonged T cell activation. Our preliminary data support the new concept that IL-27 is a critical player of the host response to Legionella infection. IL-27 is elevated in broncho-alveolar lavage fluids of human patients with Legionnaires’ disease and IL-27RA deficient mice are more resistant to infection. Here, we propose to test the central hypothesis that IL-27 is produced by Legionella infected mononuclear phagocytes and initiates dichotomous programs in lymphocytes, that include the protective activation of NK cells and adverse T cell-mediated immunosuppression in lungs. Aim 1) To pinpoint the cellular source(s) of IL-27 during Legionellosis within the subsets of lung mononuclear phagocytes. We aim to assess the contribution of relevant PRR immunosensors for IL-27 induction by L. pneumophila. Samples from Legionnaires’ disease patients will be studied for associations of IL-27 with the PD-1/PD-L1 axis and soluble markers of immunopathology. Aim 2) To characterize the functional consequences of constitutive IL-27RA ablation in infected IL-27RA-/- mice, and to study the host response programs by single-cell, multi-dimensional proteotranscriptomics (CITE-Seq/Total-Seq). We will also evaluate neutralizing IL-27 antibodies and an engineered decoy receptor to improve pneumonia severity in mice. Aim 3) Based on preliminary data, we aim to study the altered inflammatory response of our novel conditional mice with NK cell-specific deletion of IL-27RA. We will investigate whether NK cell maturation is associated with a switch of IL-27RA controlled transcriptional programs through engaging non-STAT phosphoprotein signaling. Aim 4) To investigate the roles of IL-27RA in conventional and unconventional T cells with a mechanistic focus on co-inhibitory/co-stimulatory receptors during lung infection with L....