Nlrx1-Regulated Defense and Metabolic Responses to Aspergillus fumigatus Are Morphotype and Cell Type Specific.

The Nlr family member X1 (Nlrx1) is an immuno-metabolic hub involved in mediating effective responses to virus, bacteria, fungi, cancer, and auto-immune diseases. We have previously shown that Nlrx1 is a critical regulator of immune signaling and mortality in several models of pulmonary fungal infection using the clinically relevant fungus Aspergillus fumigatus. In the absence of Nlrx1, hosts produce an enhanced Th2 response primarily by CD103+ dendritic cell populations resulting in enhanced mortality via immunopathogenesis as well as enhanced fungal burden. Here, we present our subsequent efforts showcasing loss of Nlrx1 resulting in a decreased ability of host cells to process A. fumigatus conidia in a cell-type-specific manner by BEAS-2B airway epithelial cells, alveolar macrophages, bone marrow-derived macrophages, but not bone marrow-derived neutrophils. Furthermore, loss of Nlrx1 results in a diminished ability to generate superoxide and/or generic reactive oxygen species during specific responses to fungal PAMPs, conidia, and hyphae. Analysis of glycolysis and mitochondrial function suggests that Nlrx1 is needed to appropriately shut down glycolysis in response to A. fumigatus conidia and increase glycolysis in response to hyphae in BEAS-2B cells. Blocking glycolysis and pentose phosphate pathway (PPP) via 2-DG and NADPH production through glucose-6-phosphate dehydrogenase inhibitor resulted in significantly diminished conidial processing in wild-type BEAS-2B cells to the levels of Nlrx1-deficient BEAS-2B cells. Our findings suggest a need for airway epithelial cells to generate NADPH for reactive oxygen species production in response to conidia via PPP. In context to fungal pulmonary infections, our results show that Nlrx1 plays significant roles in host defense via PPP modulation of several aspects of metabolism, particularly glycolysis, to facilitate conidia processing in addition to its critical role in regulating immune signaling.

NLRX1 is a key regulator of immune signaling during invasive pulmonary aspergillosis.

Aspergillus fumigatus is an opportunistic fungal pathogen of immunocompromised patient populations. Mortality is thought to be context-specific and occurs via both enhanced fungal growth and immunopathogenesis. NLRX1 is a negative regulator of immune signaling and metabolic pathways implicated in host responses to microbes, cancers, and autoimmune diseases. Our study indicates loss of Nlrx1 results in enhanced fungal burden, pulmonary inflammation, immune cell recruitment, and mortality across immuno-suppressed and immuno-competent models of IPA using two clinically derived isolates (AF293, CEA10). We observed that the heightened mortality is due to enhanced recruitment of CD103+ dendritic cells (DCs) that produce elevated amounts of IL-4 resulting in a detrimental Th2-mediated immune response. Adoptive transfer of Nlrx1-/- CD103+ DCs in neutropenic NRG mice results in enhanced mortality that can be ablated using IL-4 neutralizing antibodies. In vitro analysis of CD103+ DCs indicates loss of Nlrx1 results in enhanced IL-4 production via elevated activation of the JNK/JunB pathways. Interestingly, loss of Nlrx1 also results in enhanced recruitment of monocytes and neutrophils. Chimeras of irradiated Nlrx1-/- mice reconstituted with wild type bone marrow have enhanced neutrophil recruitment and survival during models of IPA. This enhanced immune cell recruitment in the absence of Nlrx1 is mediated by excessive production of CXCL8/IL-8 family of chemokines and IL-6 via early and enhanced activation of P38 in response to A. fumigatus conidia as shown in BEAS-2B airway epithelial cells. In summary, our results point strongly towards the cell-specific and contextual function of Nlrx1 during invasive pulmonary aspergillosis and may lead to novel therapeutics to reduce Th2 responses by CD103+ DCs or heightened recruitment of neutrophils.

Endocytic Markers Associated with the Internalization and Processing of Aspergillus fumigatus Conidia by BEAS-2B Cells.

Aspergillus fumigatus is a ubiquitous mold that produces small airborne conidia capable of traversing deep into the respiratory system. Recognition, processing, and clearance of A. fumigatus conidia by bronchial airway epithelial cells are thought to be relevant to host defense and immune signaling. Using z-stack confocal microscopy, we observed that only 10 to 20% of adherent conidia from the AF293 clinical isolate are internalized by BEAS-2B cells 6 h postchallenge and not prior. Similar percentages of internalization were observed for the CEA10 clinical isolate. A large subset of both AF293 and CEA10 conidia are rendered metabolically inactive without internalization at 3 h postchallenge by BEAS-2B cells. A significantly larger percentage of CEA10 conidia are metabolically active at 9 and 12 h postchallenge in comparison to the AF293 isolate, demonstrating heterogeneity among clinical isolates. We identified 7 host markers (caveolin, flotillin-2, RAB5C, RAB8B, RAB7A, 2xFYVE, and FAPP1) that consistently localized around internalized conidia 9 h postchallenge. Transient gene silencing of RAB5C, PIK3C3, and flotillin-2 resulted in a larger population of metabolically active conidia. Our findings emphasize the abundance of both host phosphatidylinositol 3-phosphate (PI3P) and PI4P around internalized conidia, as well as the importance of class III PI3P kinase for conidial processing. Therapeutic development focused on RAB5C-, PIK3C3-, and flotillin-2-mediated pathways may provide novel opportunities to modulate conidial processing and internalization. Determination of how contacted, external conidia are processed by airway epithelial cells may also provide a novel avenue to generate host-targeted therapeutics.IMPORTANCE Conidia from the fungus Aspergillus fumigatus are notorious for their ability to stay airborne. This characteristic is believed to allow conidia to penetrate into the cleanest environments. Several hundred conidia are thought to be inhaled each day by a given individual and then expelled by mucociliary clearance. Given that airway epithelial cells make up a significant portion of the pulmonary-air interface, we set out to determine the percentage of conidia that are actually internalized after initial contact with airway epithelial cells. We determined this through an in vitro assay using an immortalized bronchial airway epithelial cell line known as BEAS-2B. Our results suggest a small fraction of conidia are internalized by BEAS-2B cells, while the majority stay adherent to the surface of cells or are washed away during sample processing. Internalization of conidia was observed at 6 h postchallenge and not prior. Our data also indicate conidia are rendered metabolically inactive within 3 h of challenge, suggesting BEAS-2B cells process a large number of conidia without internalization in this early time frame. We have also identified several host endocytosis markers that localize around internalized conidia as well as contribute to the processing of conidia. Understanding how these host endocytosis markers affect the processing of internal and/or external conidia may provide a novel avenue for therapeutic development.

Modulation of Immune Signaling and Metabolism Highlights Host and Fungal Transcriptional Responses in Mouse Models of Invasive Pulmonary Aspergillosis.

Incidences of invasive pulmonary aspergillosis, an infection caused predominantly by Aspergillus fumigatus, have increased due to the growing number of immunocompromised individuals. While A. fumigatus is reliant upon deficiencies in the host to facilitate invasive disease, the distinct mechanisms that govern the host-pathogen interaction remain enigmatic, particularly in the context of distinct immune modulating therapies. To gain insights into these mechanisms, RNA-Seq technology was utilized to sequence RNA derived from lungs of 2 clinically relevant, but immunologically distinct murine models of IPA on days 2 and 3 post inoculation when infection is established and active disease present. Our findings identify notable differences in host gene expression between the chemotherapeutic and steroid models at the interface of immunity and metabolism. RT-qPCR verified model specific and nonspecific expression of 23 immune-associated genes. Deep sequencing facilitated identification of highly expressed fungal genes. We utilized sequence similarity and gene expression to categorize the A. fumigatus putative in vivo secretome. RT-qPCR suggests model specific gene expression for nine putative fungal secreted proteins. Our analysis identifies contrasting responses by the host and fungus from day 2 to 3 between the two models. These differences may help tailor the identification, development, and deployment of host- and/or fungal-targeted therapeutics.