Strain specific persistence in the murine lung of Aspergillus fumigatus conidia 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 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 not only be used to better understand disease pathogenesis of ABPA and potential anti-fungal 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 when to utilize antifungal therapies in patients with ABPA and fungal allergic type diseases.


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Individuals with atopic asthma, COPD, and particularly Cystic Fibrosis (CF) are 61 susceptible to chronic fungal colonization and infections in the lung (1). For example, 62 30-80% of CF patients persistently test positive for growth of the filamentous fungus, 63 Aspergillus fumigatus, in expectorated sputum (2-4) and this finding is associated with 64 overall decreased lung function (5). These findings demonstrate that A. fumigatus examining the fungal contribution to ABPA development, persistence, and disease 86 progression are currently difficult to conduct. This is a critical question to address 87 because it is expected to help inform when antifungal therapies may be effective in the 88 context of ABPA. 89 The goal of the current study was to identify a strain of A. fumigatus that is 90 capable of long-term persistence in the lung of immune competent mice. Using a clinical 91 strain isolated from a sputum sample, viable A. fumigatus was recovered from the 92 murine airways for up to 21 days. Animals with persistent fungal burden developed 93 5 increased serum IgE, eosinophilia, airway damage, mucus production, and an 94 increased immune response to re-exposure to fungi, all common features of ABPA. 95 Surprisingly, these symptoms developed despite little to no hyphal growth observed in 96 the airways of animals with persistent fungal burden. Consequently, these data 97 demonstrate that a fungal strain with resistance to leukocyte killing and relatively low 98 virulence is capable of long-term persistence in the murine lung and initiating ABPA-like 99 disease pathogenesis. Hygromycin-resistant and mRFP strain generation: Protoplasts from strains CEA10 and 116 W72310 were generated with Trichoderma harzianum (Millipore Sigma) lysing enzyme 117 and transformed ectopically with linear constructs of gpdA-driven hph hygromycin-118 resistance gene or H2A:mRFP (CEA10) as previously described (19,20). 119 RNA preparation and NanoString Analysis: Animals were "sensitized" to either PBS, 120 CEA10, or W72310 as described previously and lungs were removed at euthanasia for 121 mRNA analysis. Lungs were freeze dried, homogenized with glass beads using Mini     Table   230 2) and mapped against W72310 variant positions using a custom script in R. Putative   Comparison of W72310 sensitization to CEA10 sensitization in the murine lung: Given 295 the persistent presence of the W72310 strain in the murine lung, its increased 296 14 resistance to leukocyte-mediated killing, and the altered cellular response to W72310 297 conidia, we next compared the overall immune response to sensitization with CEA10 298 and W72310 live conidia. Animals were intranasally inoculated and sensitized to 299 W72310 or CEA10 conidia as indicated ( Fig. 2A). Compared to PBS-sensitized animals, 300 CEA10 and W72310 animals had 6-and 8-fold increased serum IgE, respectively (Fig.   301 2B). Although we observed a trend for more total IgE in animals exposed to W72310 302 compared to CEA10, the difference was not statistically significant. Additionally, small 303 but detectable CFUs were observed in lungs sensitized with CEA10, however, 304 approximately 10^6 CFUs were detected in lungs of animals sensitized with W72310 305 (Fig. 2C). In parallel, total RNA was extracted from whole lungs and analyzed for 306 changes in mRNA levels of immune-related genes using NanoString nCounter 307 technology. As expected, overall changes in mRNA levels were strikingly different in 308 PBS-sensitized animals compared to the two fungal strains (Fig. 2D). Increased 309 signatures in pathways related to cytokine/chemokine signaling, host pathogen 310 interactions, and innate and adaptive immune signaling were observed in fungal 311 challenged animals as expected (Fig. 2E.). Interestingly, the only immune-related 312 pathways with reduced mRNA levels in fungal-exposed mice compared to PBS were 313 TGF-β and inflammasome encoding genes (Fig. 2E). Using GO-term analysis with a 314 cut-off of P<0.01 to differentiate highly significant pathways in the W72310-sensitized 315 lung compared to PBS, analysis showed prevalent eosinophil, monocyte, and 316 lymphocyte chemotaxis, ERK1 and ERK2 signaling, IFNγ-response signaling, and 317 general immune system responses (Fig. 2F). Comparison of PBS and W72310-318 sensitized lungs revealed 174 increased and 38 decreased mRNA transcripts (Fig. 2G).

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The lack of differences between CEA10 and W72310 challenged animals seems 326 especially striking given the clear differences in persistent fungal burden.   (Fig. 4C). Conidia were strongly visible 21 days after challenge in the W72310-370 sensitized mice, however, they appeared to be taken up in large vacuolar phagocytes 371 (Fig. 4C). Interestingly, multiple conidia were visible in a single host cell. Conidia were 372 mostly non-detectable by 28 days, with a small number visible by GMS stain.

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Collectively these data demonstrate that W72310 can persist in a fungal sensitized 374 murine lung up to one month after inoculation.

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Blood was collected from each animal and total serum IgE levels were measured 376 by ELISA to determine the overall allergic response. As expected, total IgE levels 377 increased 2 and 7 days post challenge in W72310 sensitized mice compared to controls 378 (Fig. 4D), however significant increases were not detected at 21 and 28 days. 379 Interestingly, even in the PBS-sensitized animals, total serum IgE was increased at 21 380 days compared to PBS-sensitized control at 2 days. Overall, these data indicate that 381 sensitization and challenge with strain W72310 maintain high levels of IgE.

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Sensitization and Challenge with W72310 causes increased inflammation, 383 leakage, and mucous cell metaplasia: Eosinophilia is a hallmark of ABPA disease (36).

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Differential staining of airway/bronchoalveolar lavage cells revealed that eosinophils 385 were increased 2, 7, and 21 days after sensitization and challenge with W72310 live 386 conidia in comparison to PBS-sensitized controls (Fig. 5A). Moreover, macrophages 387 and lymphocytes were increased at all time points (Fig. 5A). Interestingly, lymphocytes 388 and eosinophils continued to be detectable 28 days post challenge, but at lower levels.

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Overall neutrophils decreased over time, but no significant differences were observed 390 between control and experimental groups at any time points (Fig. 5A). The inflammatory 391 response was also assessed by staining lung sections for H&E. Some cellular infiltrates 392 were observed in PBS-sensitized mice 2 and 7 days post challenge with W72310, while 393 robust inflammation, including eosinophils, was observed in animals sensitized and 394 challenged with W72310 at all time points (Fig. 5B). Furthermore, animals sensitized challenged group (Fig. 6B). Serum IgE levels were increased 5-fold in mice sensitized 412 and challenged with W72310 at 21 days compared to PBS sensitized challenged mice, 413 as would be expected (based on results from the time course (Figs. 4D)) and BAL 414 albumin was measured in order to assess damage and leakage in the airways (Fig. 6C).  (Table 1). Interestingly, no variants were identified in core metabolism genes. When  (Table 2). In order to corroborate differences observed 446 in oxidative stress resistance, mRFP-CEA10 and mRFP-W72310 strains were assayed 447 for susceptibility to oxidative stress inducing agents, hydrogen peroxide and menadione.

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The percentage of viable CEA10 conidia (RFP + ) significantly reduced by 50% (hydrogen 449 peroxide) and 40% (menadione), respectively. In contrast, incubation of W72310 with 450 hydrogen peroxide and menadione only decreased the percentage of viable conidia by 451 less than 5% (Fig. 7A). Collectively these data show that W72310 conidia are less 452 susceptible to oxidative stress-induced death compared to CEA10 and this may 453 contribute to its persistent phenotype in the mouse lung. The allele(s) driving this 454 phenotype in W72310 conidia remain to be determined in future studies. persistence of W72310 conidia in murine lungs. 468 We hypothesize that a key fungal mechanism for fungal persistence is enhanced menadione-induced death compared to CEA10 (Fig. 7) to immune cell mediated killing and had similar pathogenicity in mouse models of 481 aspergillosis compared to wild-type controls (39-41). Since there are 20 oxidative stress 482 response genes with variants in the W72310 strain, it seems likely that more than one 483 gene/mechanism is involved in its defense against ROS-induced killing. However, we 484 cannot rule out that novel alleles of genes like catA encode proteins with increased 485 activity.

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Another fungal-centric striking observation from our studies is that W72310  (Fig. 1E). LC3-associated phagocytosis has been shown to be critical in fungal killing 500 and is dependent on exposure of the fungal cell wall components, melanin and β-glucan 501 (44). Sequence analysis showed high impact SNPs in 6 cell wall genes and 6 melanin 502 genes which could potentially prevent full recognition and/or killing of the fungus (Table   503 1). Collectively, SNPs in melanin and cell wall stress genes may affect overall 504 germination and immune cell recognition of W72310 and SNPs in oxidative stress 505 response genes could affect overall viability of phagocytosed W72310 conidia. Rigorous 506 fungal genetic studies will be needed to determine specifically which gene(s) are 507 important overall for the persistent phenotype.

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Despite a similar immunological response to sensitization between W72310 and 509 CEA10, select interferon-responsive genes were differentially expressed. CXCR3 was 510 not differentially expressed between W72310 and CEA10 (data not shown), however, 511 expression of its ligands, CXCL-9 and CXCL-10, was higher in W72310-sensitized 512 lungs compared to CEA10. CXCL9 and CXCL10, which have been shown to be induced severe asthma and in humans with severe asthma, CXCL-10 was shown to play a 518 critical role in corticosteroid resistance and Th1-mediated inflammation (49). However, 519 differences in the immune response at this time point could be either in response to the 520 different strains of fungi or different quantities of fungi since we observe significantly 521 more W72310 CFUs at this time point (Fig. 2C).

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The results of our present study indicate that persistent conidial colonization in    Non-synonamous variants found in W7 relative to AF293, that are not found in CEA10 Gene ID # Exclusive Variants Annotation Afu2g01520 10 Ortholog(s) have role in ascospore formation, hyphal growth, response to oxidative stress, sporocarp development involved in sexual reproduction