Lung infection due to Streptococcus pneumoniae (Sp) is the leading cause of community acquired pneumonia and a major cause of both morbidity and mortality in the United States and worldwide. The incidence of pneumococcal pneumonia is increasing, due, in part, to the advancing age of the at-risk population. The changing epidemiology of pneumococcal pneumonia highlights the importance of more fully understanding innate immune responses to Sp and other bacterial pathogens of the lung. The IL-36 family is a group of novel IL-1-like pro-inflammatory cytokines that are highly expressed in mucosal surfaces by both epithelial cells and bone-marrow derived immune cells. Two members of the IL-36 family, IL-36? and IL-36?, have recently been shown to induce lung inflammation and the expression of inflammatory cytokines and chemokines in vivo. However, nothing is known regarding their role in innate immunity, particularly protective lung mucosal immunity. In preliminary studies, we have found that one member of the IL-36 family, IL-36?, is produced by lung macrophages in response to Sp infection. The mechanism(s) by which IL-36 family members are secreted is not well understood. Like IL-1?, IL-36 family members lack a secretory signal sequence, and our preliminary studies suggest that IL-36? may be released in plasma membrane containing microparticles and/or exosomes rather than by classical golgi-dependent secretion. Importantly, IL-36? potently skews dendritic cells (DC) to produce type 1 and IL-17 cytokines. Moreover, we have found that IL-36? blockade/deletion results in marked impairment in lung bacterial clearance, which is associated with reduced generation of type 1 and IL-17 cytokines and chemokines. These exciting preliminary studies provide the foundation for our hypothesis that IL-36 family members are important components of effective type 1- and IL-17-mediated immunity against Gram-positive bacterial pathogens of the lung. Moreover, IL-36? is secreted in microparticles and/or exosome that may maximize distribution and/or biological activity. To further understand the contribution of IL-36 family members to the innate immune responses to infection, we propose the following specific aims:
Specific Aim 1 : Determine the cellular sources and mechanisms of secretion of IL-36? in the lung in response to pneumococcal infection. Experiments in this aim will define the cellular sources of IL-36 family members in the lung during murine S. pneumoniae infection, and determine mechanisms of secretion of IL-36 family members in mouse and human lung macrophages. In particular, we will determine whether: IL-36? secretion by lung macrophages in response to Sp occurs in a non-golgi dependent fashion; requires Sp virulence factors; is released in microparticles and/or exosomes; and will identify truncated IL-36? isoforms and other relevant cytokines in IL-36? containing microparticles and/or exosomes.
Specific Aim 2 : Determine the effect of IL-36 family members on host innate lung antipneumococcal responses in-vivo and in-vitro. Experiments in this aim will seek to define the in vivo phenotype associated with IL-36? or IL-36 receptor blockade (using neutralizing antibody and genetic deletion). Specific endpoints include: lung bacterial clearance, dissemination and survival; leukocyte recruitment; dendritic cell and lung macrophage activation; and type 1 and IL-17 cytokines, chemokine, and antimicrobial peptide expression. Effects of IL-36?-mediated DC effector responses will be evaluated in-vitro.
Specific Aim 3 : Determine the effect of IL-36? biological activity or IL-36? responsiveness inmutant mice using i.t. reconstitution or DC adoptive transfer strategies. In this aim, we will employ proof-of-concept approaches to reconstitute IL-36? biological activity or responsiveness by using direct i.t. administration of IL-36?-containing microparticles, DC-derived type 1 or IL-17 cytokines, or adoptive transfer of wild-type DC or DC genetically deficient in selected cytokines. End points include bacterial clearance, cellular recruitment/activation, and cytokine/chemokine responses. The complementary strategies proposed in this application will identify a novel IL-36?-mediated pathway of protective innate type 1 and IL-17 responses in pneumococcal pneumonia.
Lung infection due to Streptococcus pneumoniae is the leading cause of community acquired pneumonia and a major cause of both morbidity and mortality in the United States and worldwide. We have identified a critical role for IL-36?, a novl member of the IL-1 cytokine family, in protective lung innate immunity against S. pneumoniae. This cytokine induces protection, in part, by stimulating dendritic cell mediated type 1 and IL-17 responses. The studies proposed in this application may identify a novel cytokine mediated pathway of protective innate immunity in bacterial pneumonia.
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