Investigating mammalian innate immune responses to pathogenic fungi

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

In FY25, our lab has made tremendous progress in several research areas related to host-fungal interactions. We have had two main focuses over the past year: 1) Understanding the mechanisms underlying fungal persistence within granulomas, 2) Understanding how secreted molecules control barrier tissue biology Area 1: Fungi are an increasing global health problem that largely infect immunocompromised patients. While iatrogenic fungal exposure is common in healthcare settings, invasive fungal disease is often a result of reactivated latent infection. Granulomas are complex immunological structures that function to physically prevent pathogen dissemination from the site of primary infection but simultaneously can support persistent latent infection. The mechanisms that control granuloma formation, and the immunological pathways that regulate fungal persistence in these structures remain undefined. In FY25, our lab has made several key advances in understanding this biology. Using a murine fungal pulmonary granuloma model (infection with Cryptococcus neoformans strain deficient in glucosylceramide synthase), we have found that deletion of type 2 cytokines (IL-4/IL-13) results in clearance of fungi from the lungs, whereas wild type mice maintain pulmonary burden up to at least 150 days. From flow cytometry and scRNA-seq experiments, we observe a large expansion of alternatively activated macrophages (AAMs) in the lungs during granulomatous infection, and deletion of these cells similarly results in fungal clearance, suggesting that the macrophage is the main target of type 2 cytokine signaling. Interestingly, while the textbook model is that AAMs represent an intracellular replication niche due to their downregulation of cell-autonomous killing machinery (ROS, iNOS, etc), in mixed bone marrow chimera experiments where we compete wild type and Stat6-deficient macrophages in the same tissue, we observe no difference in intracellular fungal burden. In parallel, we have utilized fixed and live tissue microscopy experiments to observe that most of the fungi in granulomas are extracellular, suggesting that AAMs drive fungal persistence through cell-extrinsic mechanisms. To test which cells are the dominant source of type 2 cytokines within granulomas, we infected IL-4/IL-13 reporter mice and looked at sequential time points during infection (day 10, 20, 30, 40, 50). We find that eosinophils, basophils, and Th2 cells are the major sources of IL-4, whereas Th2 and ILC2s are the dominant IL-13 producers. To test which cells are the most relevant source of type 2 cytokine, we infected mice genetically deficient in eosinophils (EPO-DTA), ILC2 (Klrg1-Cre x Gata3flox/flox), or Th2 (hCD2-Cre x Gata3flox/flox) and tested CFU. Interestingly, while ILC2 and eosinophil ablation had little effect on fungal burden, deletion of Th2s resulted in almost complete clearance of fungi from granulomas. Collectively, these data suggest that Th2-macrophage interactions are crucial for driving fungal persistence within granulomas. To understand factors that control macrophage-Th2 interactions, we performed scRNA-seq on lung macrophages during C. neoformans infection. Interestingly, the enzyme cholesterol-25-hydroxylase (Ch25h) emerged as one of the most stringent markers of infection-induced macrophages. Ch25h converts cholesterol into the oxysterol 25-hydroxycholesterol (25-HC), which can be converted into 7a,25-HC by the enzyme Cyp7b1. 7a,25-HC is a chemotactic oxysterol that functions as a ligand for the G protein-coupled receptor EBI2/GPR183, which is highly expressed by CD4 T cells. While Ch25h induction in macrophages is usually interferon-dependent, we find that it is actually IL-4/IL-13 inducible in pulmonary macrophages. Deletion of Ch25h from macrophages (CD11c-Cre x Ch25hflox/flox) results in decreased pulmonary burden and a reduction of EBI2 chemotactic activity as readout by bioassays. Reciprocally, deletion of EBI2 specifically on Th2 cells (EBI2-/-:hCD2-Cre x Gata3flox/flox mixed bone marrow chimeras), results in a striking decrease in pulmonary fungal burden, suggesting that macrophage production of oxysterol gradients locally positions Th2 cells within granulomas to supply type 2 cytokine. From EBI2 knockout competitive bone marrow chimeras, we find no difference in Th2 recruitment into the lungs, suggesting that EBI2 mainly functions in tissue positioning rather than entry. We have also performed imaging experiments utilizing EBI2-GFP/+ vs EBI2-GFP/GFP knock-in mice, and found that cells deficient in EBI2 seem to form large clusters around bronchioles, suggesting that EBI2 is required to move Th2s away from the site of entry and into the granuloma. In summary, we have identified an oxysterol-cytokine feedforward loop that maintains macrophage alternative activation in granulomas to drive fungal persistence. All of the above described work has been submitted for publication and is either currently under review or in revision. Area 2: Fungi are understudied components of our commensal intestinal microbiota. While they only comprise about 1-2% of the total species abundance in the gut, fungi are also around 10x larger than bacteria and as eukaryotes they may have specialized secretory molecules that can interface with the host. Wallemia mellicola is a commensal fungus and common food contaminant that has been linked to increased asthma sensitivity when it overgrows in the intestine. However, the mechanisms underlying this phenomenon remain unclear. Tuft cells are a specialized chemosensory epithelial cell that sense environmental metabolites to drive secretion of IL-25. IL-25 then activates ILC2s to produce IL-13, which promotes further differentiation of tuft cells from intestinal crypt stem cells. IL-25 stimulation of ILC2s can also drive their egress from the lamina propria, allowing them to seed distal organs. Interestingly, this type 2 cytokine feedforward circuit has only been described in the small intestine, and experiments in the field thus far have found colonic tuft cell differentiation to be refractory to type 2 cytokines. We have found that oral gavage with Wallemia mellicola results in specific expansion/activation of colonic tuft cells, which secrete IL-25 to drive recirculation of ILC2s from the intestinal lamina propria into the lungs. Importantly, we find that the W. mellicola-driven colonic tuft cell expansion does not require Stat6, suggesting a type 2 cytokine-independent mechanism. Utilizing in vitro colonic organoid models, we find that W. mellicola conditioned supernatants are sufficient to induce tuft cell differentiation, suggesting that the fungus produces a secreted molecule that can directly modulate intestinal stem cell differentiation. In biochemical testing, this tuft cell-inducing activity is both heat- and protease-sensitize, consistent with a secreted protein. Using ammonium sulfate precipitation and size-exclusion chromatography to fractionate these supernatants for subsequent testing of tuft cell-induction capacity in organoids followed by mass spectrometry, we have identified a secreted L-asparaginase as the functional W. mellicola-secreted protein. We have confirmed this using commercial recombinant L-asparaginase, which is sufficient to induce tuft cell differentiation in organoids and in the colon. We are now probing the molecular mechanism underlying this phenomenon, specifically asking whether asparagine depletion is a tuft cell triggering factor, or whether L-asparaginase functions to induce tuft cells via another pathway.