GM-CSF-mediated epithelial-immune cell crosstalk orchestrates pulmonary immunity to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening mold pneumonia in immune compromised patients, particularly in those with quantitative or qualitative defects in neutrophils. While innate immune cell crosstalk licenses neutrophil antifungal activity in the lung, the role of epithelial cells in this process is unknown. Here, we find that that surfactant protein C (SPC)-expressing lung epithelial cells integrate infection-induced IL-1 and type III interferon signaling to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) preferentially at local sites of fungal infection and neutrophil influx. Using in vivo models that distinguish the role of GM-CSF during acute infection from its homeostatic function in alveolar macrophage survival and surfactant catabolism, we demonstrate that epithelial-derived GM-CSF increases the accumulation and fungicidal activity of GM-CSF-responsive neutrophils, with the latter being essential for host survival. Our findings establish SPC + epithelial cells as a central player in regulating the quality and strength of neutrophil-dependent immunity against inhaled mold pathogens. HIGHLIGHTS: GM-CSF is essential for host defense against A. fumigatus in the lung IL-1 and IFN-λ promote GM-CSF production by lung epithelial cells in parallelEpithelial cell-derived GM-CSF increases neutrophil accumulation and fungal killing capacityEpithelial cells preferentially upregulate GM-CSF in local sites of inflammation.

Dectin-1/CARD9 induction of the TFEB and TFE3 gene network is dispensable for phagocyte anti-Aspergillus activity in the lung.

Myeloid phagocytes of the respiratory immune system, such as neutrophils, monocytes, and alveolar macrophages, are essential for immunity to Aspergillus fumigatus, the most common etiologic agent of mold pneumonia worldwide. Following the engulfment of A. fumigatus conidia, fusion of the phagosome with the lysosome is a critical process for killing conidia. TFEB and TFE3 are transcription factors that regulate lysosomal biogenesis under stress and are activated by inflammatory stimuli in macrophages, but it is unknown whether TFEB and TFE3 contribute to anti-Aspergillus immunity during infection. We found that lung neutrophils express TFEB and TFE3, and their target genes were upregulated during A. fumigatus lung infection. In addition, A. fumigatus infection induced nuclear accumulation of TFEB and TFE3 in macrophages in a process regulated by Dectin-1 and CARD9. Genetic deletion of Tfeb and Tfe3 impaired macrophage killing of A. fumigatus conidia. However, in a murine immune-competent Aspergillus infection model with genetic deficiency of Tfeb and Tfe3 in hematopoietic cells, we surprisingly found that lung myeloid phagocytes had no defects in conidial phagocytosis or killing. Loss of TFEB and TFE3 did not impact murine survival or clearance of A. fumigatus from the lungs. Our findings indicate that myeloid phagocytes activate TFEB and TFE3 in response to A. fumigatus, and while this pathway promotes macrophage fungicidal activity in vitro, genetic loss can be functionally compensated in the lung, resulting in no measurable defect in fungal control and host survival.

Dectin-1/CARD9-induction of the TFEB and TFE3 gene network is dispensable for phagocyte anti-Aspergillus activity in the lung.

Myeloid phagocytes of the respiratory immune system, such as neutrophils, monocytes, and alveolar macrophages, are essential for immunity to Aspergillus fumigatus, the most common etiologic agent of mold pneumonia worldwide. Following engulfment of A. fumigatus conidia, fusion of the phagosome with the lysosome, is a critical process for killing conidia. TFEB and TFE3 are transcription factors that regulate lysosomal biogenesis under stress and are activated by inflammatory stimuli in macrophages, but it is unknown whether TFEB and TFE3 contribute to anti-Aspergillus immunity during infection. We found that lung neutrophils express TFEB and TFE3, and their target genes were upregulated during A. fumigatus lung infection. Additionally, A. fumigatus infection induced nuclear accumulation of TFEB and TFE3 in macrophages in a process regulated by Dectin-1 and CARD9 signaling. Genetic deletion of Tfeb and Tfe3 impaired macrophage killing of A. fumigatus conidia. However, in a murine immune competent Aspergillus infection model with genetic deficiency of Tfeb and Tfe3 in hematopoietic cells, we surprisingly found that lung myeloid phagocytes had no defects in conidial phagocytosis or killing. Loss of TFEB and TFE3 did not impact murine survival or clearance of A. fumigatus from the lungs. Our findings indicate that myeloid phagocytes activate TFEB and TFE3 in response to A. fumigatus, and while this pathway promotes macrophage fungicidal activity in vitro, genetic loss can be functionally compensated at the portal of infection in the lung, resulting in no measurable defect in fungal control and host survival.

Gut microbiota modulates bleomycin-induced acute lung injury response in mice.

BACKGROUND: Airway instillation of bleomycin (BLM) in mice is a widely used, yet challenging, model for acute lung injury (ALI) with high variability in treatment scheme and animal outcomes among investigators. Whether the gut microbiota plays any role in the outcome of BLM-induced lung injury is currently unknown. METHODS: Intratracheal instillation of BLM into C57BL/6 mice was performed. Fecal microbiomes were analyzed by 16s rRNA amplicon and metagenomic sequencing. Germ-free mice conventionalization and fecal microbiota transfer between SPF mice were performed to determine dominant commensal species that are associated with more severe BLM response. Further, lungs and gut draining lymph nodes of the mice were analyzed by flow cytometry to define immunophenotypes associated with the BLM-sensitive microbiome. RESULTS: Mice from two SPF barrier facilities at the University of Chicago exhibited significantly different mortality and weight loss during BLM-induced lung injury. Conventionalizing germ-free mice with SPF microbiota from two different housing facilities recapitulated the respective donors' response to BLM. Fecal microbiota transfer from the facility where the mice had worse mortality into the mice in the facility with more survival rendered recipient mice more susceptible to BLM-induced weight loss in a dominant negative manner. BLM-sensitive phenotype was associated with the presence of Helicobacter and Desulfovibrio in the gut, decreased Th17-neutrophil axis during steady state, and augmented lung neutrophil accumulation during the acute phase of the injury response. CONCLUSION: The composition of gut microbiota has significant impact on BLM-induced wasting and death suggesting a role of the lung-gut axis in lung injury.

Aspergillus fumigatus Strain-Specific Conidia Lung Persistence Causes an Allergic Broncho-Pulmonary Aspergillosis-Like Disease Phenotype.

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic broncho-pulmonary aspergillosis (ABPA) is a disease diagnosed primarily in cystic fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310, which was isolated from the expectorated sputum of an ABPA patient, by whole-genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide, and menadione, in vitro In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease.IMPORTANCE Allergic broncho-pulmonary aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can be used not only to better understand disease pathogenesis of ABPA and potential antifungal treatments but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question of when to utilize antifungal therapies in patients with ABPA and fungal allergic-type diseases.

During Aspergillus Infection, Monocyte-Derived DCs, Neutrophils, and Plasmacytoid DCs Enhance Innate Immune Defense through CXCR3-Dependent Crosstalk.

Aspergillus fumigatus, a ubiquitous mold, is a common cause of invasive aspergillosis (IA) in immunocompromised patients. Host defense against IA relies on lung-infiltrating neutrophils and monocyte-derived dendritic cells (Mo-DCs). Here, we demonstrate that plasmacytoid dendritic cells (pDCs), which are prototypically antiviral cells, participate in innate immune crosstalk underlying mucosal antifungal immunity. Aspergillus-infected murine Mo-DCs and neutrophils recruited pDCs to the lung by releasing the CXCR3 ligands, CXCL9 and CXCL10, in a Dectin-1 and Card9- and type I and III interferon signaling-dependent manner, respectively. During aspergillosis, circulating pDCs entered the lung in response to CXCR3-dependent signals. Via targeted pDC ablation, we found that pDCs were essential for host defense in the presence of normal neutrophil and Mo-DC numbers. Although interactions between pDC and fungal cells were not detected, pDCs regulated neutrophil NADPH oxidase activity and conidial killing. Thus, pDCs act as positive feedback amplifiers of neutrophil effector activity against inhaled mold conidia.

Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment.

The inbred mouse strain C57BL/6J is widely used in models of immunological and infectious diseases. Here we show that C57BL/6J mice have a defect in neutrophil recruitment to a range of inflammatory stimuli compared with the related C57BL/6N substrain. This immune perturbation is associated with a missense mutation in Nlrp12 in C57BL/6J mice. Both C57BL/6J and NLRP12-deficient mice have increased susceptibility to bacterial infection that correlates with defective neutrophil migration. C57BL/6J and NLRP12-deficient macrophages have impaired CXCL1 production and the neutrophil defect observed in C57BL/6J and NLRP12-deficient mice is rescued by restoration of macrophage NLRP12. These results demonstrate that C57BL/6J mice have a functional defect in NLRP12 and that macrophages require NLRP12 expression for effective recruitment of neutrophils to inflammatory sites.