Biology subjects

Rosowski, E. E.

Publications and source records attributed to Rosowski, E. E..

6 recordsLinked to original sources

bioRxiv2026

Macrophage PHOX Restricts Aspergillus fumigatus Germination and Hyphal Development Through ROS-Dependent Immunity

Aspergillus fumigatus is a fungal pathogen that causes invasive disease in immunocompromised individuals. Innate immune cells, including macrophages and neutrophils, are essential for controlling A. fumigatus, but the mechanisms by which specific phagocytes control distinct stages of infection remain incompletely understood. In particular, the contribution of macrophage-derived reactive oxygen species (ROS) to conidial clearance and control of fungal germination and hyphae remains unclear. Here, we used larval zebrafish with mutation of p22, an essential subunit of the phagocyte NADPH oxidase (PHOX) complex, and neutrophil-deficiency, to define the role of macrophage-derived ROS during A. fumigatus infection. Mutation of p22 or pharmacological ROS scavenging increased host susceptibility, supporting a role for ROS in macrophage-mediated defense. However, macrophage ROS restricts fungal developmental progression rather than promoting clearance of conidia. Using {Delta}pksP conidia, we confirmed that fungal melanin limits the localization of p22 to phagosomes, but found no difference in conidial killing of {Delta}pksP conidia, supporting the finding that PHOX-generated ROS are not required for macrophage conidial killing. However, {Delta}pksP conidia have defective virulence only in ROS-producing larvae, as melanin inhibits ROS-mediated control of germination and hyphal growth. These findings identify macrophage PHOX activity as a stage-specific regulator of A. fumigatus infection that primarily restricts fungal developmental progression, while PksP-produced melanin promotes immune evasion by limiting macrophage ROS-dependent antifungal activity. These findings provide insight into how macrophages inhibit the transition from conidia to invasive fungal growth and how A. fumigatus evades this defense, with implications for understanding determinants of disease progression during invasive aspergillosis.

immunology↗

bioRxiv2025

Macrophage vacuolar ATPase (v-ATPase) function controls Aspergillus fumigatus germination and hyphal growth independent of spore killing

Tissue-resident macrophages efficiently internalize Aspergillus fumigatus spores, forming a critical first line of defense against infection. However, the mechanisms that these cells use to control spores in vivo remain incompletely defined. Here, we used the live imaging capabilities of the larval zebrafish host model to assess the role of the v-ATPase complex in macrophage-mediated defense against A. fumigatus in a whole vertebrate animal. For the first time we are able to visualize co-localization of A. fumigatus spores with the key v-ATPase subunit Atp6v1h in macrophages inside of an infected animal. As macrophages only have a low ability to kill spores, this co-localization occurs as early as 1-day post-injection and persists for multiple days. Surprisingly, macrophage spore killing is not further reduced by targeting of atp6v1h with CRISPR/Cas9. Instead, v-ATPase deficiency profoundly impacts macrophage-mediated control of spore swelling, decoupling the macrophage functions of spore killing and inhibition of germination. We also identify a role for the v-ATPase complex in macrophage control of extracellular hyphal growth. These effects on macrophage function drive significantly decreased host survival in larvae lacking a functional v-ATPase. We also report broad effects of v-ATPase deficiency on macrophage numbers, apoptosis in the hematopoietic tissue, and potential neutrophil functions, reflecting the importance of this complex in host antifungal immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/664761v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d494f2org.highwire.dtl.DTLVardef@7ecc81org.highwire.dtl.DTLVardef@a45584org.highwire.dtl.DTLVardef@ea5cd3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIv-ATPase function in macrophages restricts Aspergillus germination and hyphal growth C_LIO_LISpore killing by macrophages is independent of v-ATPase function C_LIO_LILoss of macrophage v-ATPase mimics the effects of macrophage deficiency in vivo C_LI

immunology↗

bioRxiv2024

Zebrafish use conserved CLR and TLR signaling pathways to respond to fungal PAMPs in zymosan

Pattern recognition receptors (PRRs) such as C-type lectin receptors (CLRs) and Toll-like receptors (TLRs) are used by hosts to recognize pathogen-associated molecular patterns (PAMPs) in microorganisms and to initiate innate immune responses. While PRRs exist across invertebrate and vertebrate species, the functional homology of many of these receptors is still unclear. In this study, we investigate the innate immune response of zebrafish larvae to zymosan, a {beta}-glucan-containing particle derived from fungal cell walls. Macrophages and neutrophils robustly respond to zymosan and are required for zymosan-induced activation of the NF-{kappa}B transcription factor. Full activation of NF-{kappa}B in response to zymosan depends on Card9/Syk and Myd88, conserved CLR and TLR adaptor proteins, respectively. Two putative CLRs, Clec4c and Sclra, are both required for maximal sensing of zymosan and NF-{kappa}B activation. Altogether, we identify conserved PRRs and PRR signaling pathways in larval zebrafish that promote recognition of fungal PAMPs. These results inform modeling of human fungal infections in zebrafish and increase our knowledge of the evolution and conservation of PRR pathways in vertebrates.

immunology↗

bioRxiv2023

Glucocorticoids suppress NF-κB-mediated neutrophil control of Aspergillus fumigatus hyphal growth

Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus. Prolonged glucocorticoid therapy is one of the main risks for invasive aspergillosis, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. Here, we used a zebrafish larva-Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to the infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function, rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. One pathway that glucocorticoids can inhibit is NF-{kappa}B activation and we report that dexamethasone partially suppresses NF-{kappa}B activation at the infection site by inducing the transcription of I{kappa}B via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKK{gamma} to prevent NF-{kappa}B activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKK{gamma} crispant larvae further increases invasive hyphal growth, suggesting that dexamethasone may suppress other pathways in addition to NF-{kappa}B to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-{kappa}B-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae. Author SummaryGlucocorticoids are drugs that stop inflammation and suppress the immune system. Glucocorticoids are effective in treating inflammatory diseases such as asthma and arthritis, preventing organ rejection after transplant surgery, and in ameliorating the side effects of cancer chemotherapy. However, as these drugs suppress the immune system, patients taking glucocorticoids are more prone to infections such as with the environmental fungus Aspergillus fumigatus. The specific mechanisms that glucocorticoids inhibit to increase susceptibility to infection are largely unknown. Here, we used a larval zebrafish model of A. fumigatus infection to determine that glucocorticoids mainly suppress the ability of neutrophils to control the fungal hyphal growth that causes tissue damage. Our study provides insight into future strategies to treat A. fumigatus infection in patients undergoing glucocorticoid therapy.

immunology↗

bioRxiv2019

Cell type specific gene expression profiling reveals a role for the complement component C3A in neutrophil migration to tissue damage

Following acute injury, leukocytes rapidly infiltrate into tissues. For efficient recruitment, leukocytes must sense and respond to signals from both from the damaged tissue and from one another. However, the cell type specific transcriptional changes that influence leukocyte recruitment and wound healing have not been well characterized. In this study, we performed a large-scale translating ribosome affinity purification (TRAP) and RNA sequencing screen in larval zebrafish to identify genes differentially expressed by neutrophils, macrophages, and epithelial cells in the context of wounding. We identified the complement pathway and c3a.1, homologous to the C3A component of human complement, as significantly increased in neutrophils in response to a wound. We report that c3a.1-/- zebrafish larvae have impaired neutrophil responses to both tail wounds and localized bacterial infections, as well as increased susceptibility to infection due to a neutrophil-intrinsic function of C3A. We further show that C3A enhances migration of human primary neutrophils to IL-8 and that c3a.1-/- larvae have impaired neutrophil migration in vivo, and a decrease in neutrophil directed migration speed early after wounding. Together, our findings suggest a role for C3A in mediating efficient neutrophil migration to damaged tissues and support the power of TRAP to identify cell-specific changes in gene expression associated with wound-associated inflammation.

immunology↗

bioRxiv2019

Imaging invasive fungal growth and inflammation in NADPH oxidase-deficient zebrafish

Neutrophils are primary cells of the innate immune system that generate reactive oxygen species (ROS) and mediate host defense. Deficient phagocyte NADPH oxidase (PHOX) function leads to chronic granulomatous disease (CGD) that is characterized by invasive infections including those by the generally non-pathogenic fungus Aspergillus nidulans. The role of neutrophil ROS in this specific host-pathogen interaction remains unclear. Here, we exploit the optical transparency of zebrafish to image the effects of neutrophil ROS on invasive fungal growth and neutrophil behavior in response to Aspergillus nidulans. In a wild-type host, A. nidulans germinates rapidly and elicits a robust inflammatory response with efficient fungal clearance. PHOX-deficient larvae have increased susceptibility to invasive A. nidulans infection despite robust neutrophil infiltration. Expression of p22phox specifically in neutrophils does not affect fungal germination but instead limits the area of fungal growth and excessive neutrophil inflammation and is sufficient to restore host survival in p22phox-deficient larvae. These findings suggest that neutrophil ROS limits invasive fungal growth and has immunomodulatory activities that contribute to the specific susceptibility of PHOX-deficient hosts to invasive A. nidulans infection.

cell biology↗