Role of the Trophic Forms of Pneumocystis in Lung Response to Infection

Pneumocystis jirovecii, a harbinger of the HIV epidemic, causes pneumonia in immunosuppressed individuals {Friedman-Kien, 1981 #3743;Gottlieb, 1981 #3744;Gottlieb, 1981 #2546}. It has been more recently appreciated that Pneumocystis (PC) is associated with chronic obstructive pulmonary disease and asthma in HIV-infected individuals {Morris, 2004 #1529;Gingo, 2012 #4001}. The fungal pathogen has been difficult to study over the years as there is no reliable, continuous culture system making genetic manipulation impossible at this time. There are two identifiable life forms of PC, an ascus form (also called a cyst) that can have up to 8 spores inside, and the trophic form that lacks a fungal cell wall. In infected lungs, the trophic forms represent as much as 90% of the organism burden. The asci are likely the transmitted forms of the organisms and are characterized by having â-1,3 and â-1,6- rich cell walls {Kottom, 2015 #3924}. â-glucans of the asci interact with â-glucan receptors on macrophages, dendritic cells (DCs), and lung epithelial cells to stimulate cytokine production {Carmona, 2006 #1312;Evans, 2005 #2379;Hahn, 2003 #1149;Lebron, 2003 #2796}. Mannosylated proteins on both life forms interact with pattern recognition receptors such as mannose receptor (MR) and dectin-2 to stimulate phagocytosis and cytokine production {Ezekowitz, 1991 #164;Kottom TJ, 2018 #3973}. For many years the immune response to PC was studied using all of the life forms found in the lungs. There is a gap in knowledge regarding the contributions of the two life PC forms to induction of the lung immune response. Recently, we found that the trophic forms of PC induce a significantly different response in bone marrow-derived dendritic cells (BMDCs) in vitro than do the ascus form. Stimulation of BMDCs with organisms isolated from the lungs of infected Rag-/- mice results in production of TNF {Evans, 2016 #3820;Evans, 2017 #3965}. However, isolated trophic forms failed to induce TNF production and in fact inhibited TNF production by BMDCs stimulated with purified â-glucans, lipopolysaccharide (LPS), or lipoteichoic acid (LTA) {Evans, 2016 #3820;Evans, 2017 #3965}. It is currently unclear what the significance of this observation is for the lifestyle of the organisms in the lungs. Data from several labs and confirmed by us indicates that treatment of PC infected mice with a â-glucan synthase inhibitor (echinocandin class of drug) allows us to isolate trophic forms in vivo and gives us a tool for examining the differential immune responses to the different life forms of PC {Nollstadt, 1994 #4013;Schmatz, 1990 #4012;Evans, 2017 #3966;Cushion, 2010 #1801;Cushion, 2018 #4000}. Utilization of echinocandins in mice allows us to differentiate the contributions of the trophic and ascus forms of PC to induction of lung inflammation and lung injury. Our preliminary data shows that PC-infected echinocandin-treated mice have significantly reduced TNF in the lungs after a low dose stimulation with LPS. Based on these data we hypothesize that the trophic forms of PC act to temper the inflammatory response to â-glucans in the ascus forms of PC as well as co-infectious agents. The goal of this proposal is to determine the significance of the inhibitory activity of trophic forms of PC in vivo. Aim 1 Determine whether trophic forms of PC alter the balance of inflammation induced by â-glucan in vivo. A. Mice will be challenged with â-glucans in the presense of the trophic forms of PC to determine whether trophic forms modulate inflammation induced by the ascus forms of PC. B. Knockout mice or blocking antibodies will be utilized to explore potential mechanisms of interactions of trophic forms with innate immune cells. Aim 2 Determine whether trophic forms of PC dampen inflammation induced by innate or adaptive immune responses. A. In vivo models of co-infection with whole PC or trophic life forms will be used to determine whether trophic forms have activity that prevents or reduces inflammation and lung injury induced by the innate response to antigen. B. In vivo models of co-infection will be used to determine whether trophic forms of PC prevent or reduce inflammation and lung injury induced by adaptive immune response to antigen. Together, these aims will address whether trophic forms of PC alter the immune response to â-glucans as a model of the ascus forms of PC. Moreover, we will determine whether the trophic forms of PC are able to have a generalized effect on lung inflammation caused by viral and bacterial products.