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      Roles of Host Phospholipase D during Aspergillus fumigatus Infection in Mice

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            Abstract

            Objective:

            Aspergillus fumigatus infection in the lungs is accompanied by the recruitment of innate immune cells, phagocytosis, and the release of inflammatory factors. Phospholipase D (PLD) is a key regulator of cell migration and phagocytosis, but the effect of PLD deficiency on antifungal infection in animals is unknown. This study aims to investigate the impact of PLD on the host immune response to A. fumigatus infection under either immunocompetent or immunosuppressed status.

            Methods:

            The invasive pulmonary aspergillosis mouse model was created using a modified protocol with immunosuppression by steroids. For collection of bronchoalveolar lavage fluid (BALF) from mice, the lungs were washed eight times with 0.5 ml of PBS. Total cell counts in BALF were determined using a Coulter Counter. The content of alveolar macrophages, neutrophils, and monocytes in BALF was examined by flow cytometry and analyzed by FlowJo V10 software. Multiplex immunoassays were used to determine the concentrations of inflammatory cytokines in BALF.

            Results:

            In immunocompetent mice, alveolar macrophages were the major cell population in BALF after A. fumigatus infection, and a number of neutrophils and monocytes were recruited in the alveoli. Loss of both pld1 and pld2 genes did not affect the content of alveolar macrophages, neutrophils, or monocytes in BALF. Under immunosuppression induced by hydrocortisone acetate, pld1-/-pld2-/- mice showed higher mortality after A. fumigatus infection and had a higher fungal burden and much lower number of prominent focal areas of dense inflammatory infiltrates in lung tissue than wild type mice. Moreover, interleukin (IL)-12p40 significantly decreased, and IL-10 markedly increased, in BALF from pld1 -/- pld2 -/- mice after infection.

            Conclusion:

            Our findings revealed that, during A. fumigatus infection, deficiency in both pld1 and pld2 in mice was not conducive to the infiltration of inflammatory cells into lung tissue but promoted the release of IL-10 and blocked the release of IL-12, thereby increasing fungal burden and mortality.

            Main article text

            INTRODUCTION

            Aspergillus fumigatus is widely found in the environment, particularly in the air, water, plants, and soil, and is the main pathogen causing aspergillosis; A. fumigatus can be isolated from more than 80% of samples from patients with aspergillosis [1]. Conidia of A. fumigatus, owing to their small size (diameter of 2–3 μm), are inhaled by humans and animals and travel deep into the lower respiratory tract; these conidia may be responsible for a variety of clinical cases. However, invasive aspergillosis, which is found primarily in immunocompromised people, remains the most dangerous because of its high mortality rates, which range from 30% to 100% [24].

            Alveoli are the principal origin of systemic infections of Aspergillus, although infection may also arise from other anatomical sites, such as the sinuses. Alveolar macrophages represent 90% of the resident leucocytes in the lungs, and they home to areas just beneath the alveolar surfactant film [5]. The alveolar macrophages engulf A. fumigatus conidia and subsequently produce cytokines and chemokines, such as TNF-α, IL-12, IFN-γ, IL-18, IL-6, IL-1β, MIP-1, MIP-1α, MIP-2, G-CSF, and GM-CSF [6]. Consequently, professional phagocytes, such as circulating macrophages, neutrophils, monocytes, and dendritic cells, are massively recruited to infection sites. These professional phagocytes play essential roles in host defense against fungal infection through various mechanisms, such as efficiently binding and ingesting A. fumigatus conidia, secreting proinflammatory cytokines, and producing reactive oxygen species [7,8]. Furthermore, damaged tissues release chemokines during inflammation, which attract white blood cells to injury sites [9,10].

            Phospholipase D (PLD) is an important phospholipase that catalyzes the hydrolysis of phosphatidylcholine, the most abundant membrane phospholipid, thus producing phosphatidic acid and choline [11]. In mammals, two PLD isoforms, PLD1 and PLD2, are found on the cell membrane surface and are responsible for the phosphatidylcholine-hydrolyzing activity [12]. Host PLD1 and PLD2 are central signaling proteins, and their deficiency in mice leads to complicated developmental, behavioral, and immunological effects, such as defects in T cell signaling and expansion, Fc-mediated phagocytosis in macrophages, and activation of NADPH oxidase in neutrophils [13]. Previous studies have shown that PLD is activated in early stages of β2 integrin-mediated phagocytosis in neutrophils [14], and the two isoforms of PLD—PLD1 and PLD2—coordinately regulate macrophage phagocytosis [15,16] and cell migration [17].

            The activity of PLD enzymes in host cells is associated with antimicrobial activities [18]. PLD enzymes in the host can be targeted to halt viral replication during infection. PLDs in macrophages participate in the phagocytosis of Yersinia pseudo tuberculosis and the elimination of Salmonella typhimurium. PLD inhibitors such as VU0285655 block the proliferation of Plasmodium falciparum (a pathogenic factor in malaria) and that of Toxoplasma gondii (the pathogenic factor in toxoplasmosis), on the basis of in vitro experiments. According to our previous research, PLD in lung epithelial cells is activated not only by β-1,3-glucan on the surfaces of A. fumigatus conidia but also by gliotoxin, the most potent toxin produced by A. fumigatus. The activation of both PLD1 and PLD2, two subtypes of mammalian PLD, is important for the efficient internalization of A. fumigatus into lung epithelial cells in vitro [19,20]. However, little is known regarding the consequences of PLD deficiency in the host (through deletion of both the pld1 and pld2 genes) on antifungal ability during A. fumigatus infection in animal models.

            In this study, we used pld1-/-pld2-/- mice to analyze the function of PLD during A. fumigatus infection under immunocompetent or immunosuppressed conditions. Deficiency in both pld1 and pld2 genes in mice had only moderate effects on the number of innate immune cells in BALF. Under immunosuppression, PLD deficiency inhibited the production of the cytokine IL-12 and increased production of the anti-inflammatory cytokine IL-10. This response was associated with an increase in A. fumigatus invasion and growth in lung tissue, and faster death in pld1-/-pld2-/- mice than wild type mice.

            MATERIALS AND METHODS

            A. fumigatus strains and culture

            The A. fumigatus wild type strain B5233 was a kind gift from Dr. K. J. Kwon-Chung (National Institutes of Health, Bethesda, Maryland). Conidia were inoculated on Sabouraud dextrose agar and propagated for 5–8 days at 37°C. Then the newly growing conidia were collected with 0.1% Tween-20 and passed through a 40 μm filter to remove hyphal fragments. Finally, the conidia were washed three times in PBS and counted with a hemocytometer before being stored at 4°C.

            Mouse infections

            All animal studies were approved by the Laboratory Animal Welfare and Ethics Committee (IACUC-13-2016-002) and were conducted according to relevant guidelines and regulations for laboratory animals. C57BL/6 mice were obtained from Fengtai Animal Center of Military Medical Science Academy. pld1-/-pld2-/- mice were obtained from Prof. Dr. Bernhard Nieswandt (University Hospital Wurzburg, Germany). During the normal feeding process, the hair, activity, mental state, balance, and basic vitality pld1-/-pld2-/- mice were comparable to those of wild type mice, and the weights of mice at 10 weeks of age did not significantly differ. With increased age, pld1-/-pld2-/- mice generally became heavier than wild type mice. The invasive pulmonary aspergillosis mouse model was created through modified immunosuppression with steroids, according to a protocol described by Sugui et al. [21]. The dosage and administration of hydrocortisone acetate were as described by Zhang et al. [22]. Image-Pro Express 6.0 was used to process the images (Media Cybernetics Inc., MA, USA). The lungs were homogenized, the homogenates were cultured on Sabouraud dextrose agar medium, and the numbers of colonies were counted after 18 h of culture.

            Collection of BALF and alveolar cells

            BALF was collected after mice were anesthetized with 25% (v/v) ethyl carbamate intraperitoneally. The lungs were washed eight times with 0.5 ml of PBS, as previously described [6]. The supernatant was frozen at −80°C for subsequent use for the quantification of inflammatory cytokines. All BALF was collected in 50 ml centrifuge tubes and centrifuged for 8 min at 500 g, 4°C. Red blood cells in cell pellets were lysed with erythrocyte lysis buffer. For subsequent flow cytometry, total cell counts in BALF were determined with a Coulter Counter (Cellometer Auto 2000).

            Flow cytometry assays

            Total cells were collected from BALF in mice and incubated with Fc block anti-mouse CD16/CD32 (Cat:101319), followed by anti-mouse antibodies, including anti-Ly6G-PE (Cat:127608), anti-CD11b-FITC (Cat:101205), and anti-F4/80-PE (Cat:123110), all from BioLegend. Total cells were examined with a BD FACSCanto II flow cytometer and analyzed by FlowJo V10 software.

            Multiplex immunoassay for inflammatory cytokines

            As previously described, the BALF supernatant was collected from mice at the indicated time points. Multiplex immunoassays were used to determine the concentrations of inflammatory cytokines in the BALF, according to the manufacturer’s instructions (eBioscience).

            Statistical analysis

            Data are presented as mean ± standard error of the mean for three independent replicates. The significance of differences was assessed with log-rank analysis (for comparative survival), one-way ANOVA, or unpaired Student’s t-tests with a 95% confidence interval in GraphPad Prism software. *p < 0.05, **p < 0.01, and ***p < 0.001.

            RESULTS

            Deletion of pld1 and pld2 genes increases death and pulmonary fungal burden after A. fumigatus infection in mice with hydrocortisone acetate induced immunosuppression. A. fumigatus is an airborne pathogen that causes life-threatening conditions in patients receiving immunosuppressive therapy or people with congenital neutrophil dysfunction [23]. Immunocompetent mice and pld1-/-pld2-/- mice have been reported not to die after A. fumigatus infection [24]. The immunosuppressed mouse model for the development of invasive pulmonary aspergillosis after A. fumigatus infection was established in the current study with hydrocortisone acetate [21]. The survival rate of pld1-/-pld2-/- mice with A. fumigatus infection was lower than that of wild type mice after immunosuppression with hydrocortisone acetate (Fig 1A); however, loss of pld1 and pld2 genes did not affect the survival rate of immunocompetent mice. In terms of immunosuppression, pld1-/-pld2-/- mice had significantly higher pulmonary fungal burden (Fig 1B), faster growth and invasion of A. fumigatus conidia in lung tissue, and more severe lung tissue damage than wild type mice. Microscopic examination of the lungs of both genotypes of mice after A. fumigatus infection revealed large areas of bronchopneumonia consisting of hyphae surrounded by extensive inflammatory infiltrates, and the pulmonary architecture was effaced by zones of necrosis and inflammation with hemorrhage and edema (Fig 1C). Greater numbers of prominent focal areas of dense inflammatory infiltrates were observed in the lungs of wild type mice than pld1-/-pld2-/- mice; moreover, the necrosis, hemorrhage, and edema in the lung tissue was more severe in pld1-/-pld2-/- mice than wild type mice, and the hyphae in the lung tissue were longer in pld1-/-pld2-/- mice than wild type mice at the same time point after infection (Fig 1C). These data suggested that loss of both pld1 and pld2 genes made immunosuppressed mice more susceptible to A. fumigatus infection, but had less of an effect on the survival rate of immunocompetent mice.

            FIGURE 1 |

            After hydrocortisone acetate induced-immunosuppression, pld1-/-pld2-/- mice die earlier and have higher pulmonary fungal burden than wild type mice after A. fumigatus infection.

            Intranasally, the mice were inoculated with 5×106 CFU conidia of A. fumigatus wild type B5233 after being immunosuppressed with hydrocortisone acetate. The immunocompetent mice were inoculated intranasally with 1×107 CFU conidia. (A) For survival analysis, ten mice were weighed every 24 h from the day of infection and were visually inspected twice daily. (B) Mice were sacrificed at post-infection day 0 (control, three mice), day 1 (five mice), day 2 (four mice), day 3 (three mice), and day 7 (three mice). Fungal burden in the lung tissue was measured by counting CFUs. (C) Lung tissues were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS), and observed by light microscopy (Olympus BX51) at a magnification of 200× or 400×. Scale bar = 50 μm. Black arrows indicate hyphae (red). The images were later processed in Image-Pro Express 6.0 (Media Cybernetics Inc., MA, USA). Three independent experiments with three individual replicates were performed. The significance of differences was assessed with log-rank analysis (for comparative survival) or unpaired Student’s t-tests with a 95% confidence interval in GraphPad Prism 8.0.2 software. * p < 0.05, ** p < 0.01.

            Effects of deficiency in both pld1 and pld2 genes on innate immune cell populations in BALF from immunocompetent mice infected by A. fumigatus. Professional phagocytes, such as macrophages, neutrophils, and monocytes, play important roles in preventing fungal infection [5]. Phagocytosis and bactericidal function in these phagocytes are inhibited in vitro when PLD function is deficient [15,25,26]. However, the effect of PLD deficiency on professional phagocytes during A. fumigatus infection was unclear. Here, the cell populations in the BALF from mice were analyzed by flow cytometry with F4/80, CD11b, and Ly6G, which are surface markers of macrophages, monocytes, and neutrophils, respectively.

            Alveolar macrophages (F4/80+CD11b-) were the predominant cell populations in BALF from immunocompetent mice before intranasal administration of A. fumigatus conidia. The proportion of alveolar macrophages in pld1-/-pld2-/- mice (93.5%) was not significantly less than that (95.9%) in wild type mice (Fig 2A). At 6 h after administration of conidia, alveolar macrophages remained the major cell populations in BALF from both wild type and pld1-/-pld2-/- mice (Fig 2B). From 0 h to 6 h, the proportions of monocytes (F4/80+CD11b+, approximately seven fold increase, from 0.28% to 1.97%) and neutrophils (Ly6G+CD11b+, approximately three fold increase, from 0.58% to 1.72%) increased significantly in wild type mice, whereas the proportions of neutrophils and monocytes in pld1-/-pld2-/- mice decreased slightly (Fig 2A, 2B). The proportion of neutrophils and monocytes in alveolar lavage fluid from wild type mice was 2.1 times and 1.9 times higher than that from pld1-/-pld2-/- mice, respectively (Fig 2B). At 24 h after conidial administration, alveolar macrophages remained the major cell population in BALF from both wild type and pld1-/-pld2-/- mice (Fig 2C). From 0 h to 24 h, the proportion of alveolar macrophages in wild type mice decreased from 95.9% to 88.2%, and the proportion of alveolar macrophages in pld1-/-pld2-/- mice decreased from 93.5% to 87.7%; however, the proportion of monocytes and neutrophils increased from 0.28% to 4.49% and from 0.58% to 5.18% in wild type mice and pld1-/-pld2-/- mice, respectively (Fig 2A, 2C). Notably, the proportions of the three types of cells were higher in wild type mice than pld1-/-pld2-/- mice (Fig 2C). Moreover, the numbers of neutrophils and monocytes in BALF gradually significantly increased after infection, whereas the number of total cells and macrophages did not change significantly in both genotypes of mice. The numbers of total cells and the three types of immune cells in the pld1-/-pld2-/- mice were slightly lower than those in the wild type mice, but no significant difference was observed between groups (Fig 2D).

            FIGURE 2 |

            In immunocompetent mice, alveolar macrophages are the major cell populations in BALF after intranasal administration of A. fumigatus conidia.

            Immunocompetent mice were inoculated intranasally with 1×107 CFU conidia and anesthetized with 25% (v/v) ethyl carbamate intraperitoneally. BALF was collected at 0 h, 6 h, and 24 h after administration. Each group in each experiment included seven mice. The alveolar lavage fluid of the seven mice was mixed in one 50 ml sterile centrifuge tube. Total cells were collected after centrifugation and incubated with Fc block anti-mouse CD16/CD32 (Cat:101319), followed by anti-mouse antibodies, including anti-Ly6G-PE (Cat:127608), anti-CD11b-FITC (Cat:101205), and anti-F4/80-PE (Cat:123110). (A–C) The content of alveolar macrophages, neutrophils and monocytes in BALF at post-administration 0 h, 6 h and 24 h was examined by BD FACSCanto II flow cytometer and analyzed by FlowJo V10 software. (D) The number of total cells in BALF were determined using a Coulter Counter (Cellometer Auto 2000), and the number of alveolar macrophages, neutrophils and monocytes in BALF was calculated based on total cell number and their respective proportions. Note: The total cells were mixed cells from the alveolar lavage fluid of seven mice in each group. Two independent experiments were performed. p < 0.05, * infected vs. uninfected mice. Abbreviations: AM, alveolar macrophages; Neu, neutrophils; Mo, monocytes.

            These findings suggested that macrophages are the major cells in the pulmonary alveoli initially after A. fumigatus infection, and neutrophils and monocytes were recruited into the pulmonary alveoli in response to A. fumigatus infection in immunocompetent mice, but knocking out both pld genes had little effect on the observed cellular recruitment.

            Effects of deficiency in both pld1 and pld2 genes on innate immune cell populations in BALF from immunosuppressed mice infected by A. fumigatus. Next, we explored the possible effects of deletion of both pld1 and pld2 genes in immunosuppressed mice on the recruitment of major innate immune cells in the lungs without A. fumigatus infection. The mice were treated with hydrocortisone acetate to establish the immunosuppression model [21]. Alveolar macrophages remained the major cell populations in BALF from both wild type and pld1-/-pld2-/- mice under immunosuppression, but their proportion was lower than that in immunocompetent mice (Fig 3A, compared with Fig 2A). At day 1 post-infection, in BALF from both wild type and pld1-/-pld2-/- mice, the proportion of alveolar macrophages descended sharply, from 76.2% to 0.26% and from 77.9% to 0.58%, respectively; however, neutrophils increased markedly, from 0.29% to 87.0% and from 0.77% to 81.4%, respectively, and became the major cell populations in BALF from both wild type and pld1-/-pld2-/- mice (Fig 3A, 3B and 3D). A consistent decrease in both the proportion (days 1 and 3) and number of neutrophils (day 1) was observed in the pld1-/-pld2-/- mice, as compared with wild type mice (Fig 3B3D); however, these differences were not significant. In addition, no statistical difference in the number of total cells and the three types of immune cells in BALF was found between groups (Fig 3D).

            FIGURE 3 |

            In mice with hydrocortisone acetate-induced immunosuppression, many neutrophils are recruited into BALF and become the major cell populations during A. fumigatus infection.

            Mice with immunosuppression were inoculated intranasally with 5×106 CFU conidia and anesthetized by intraperitoneal administration of 25% (v/v) ethyl carbamate. BALF was collected at post-infection day 0, day 1, and day 3. The number of mice in each group ranged from five to seven in each experiment. The alveolar lavage fluid of each group was mixed in one 50 ml sterile centrifuge tube. Total cells were collected after centrifugation and incubated with Fc block anti-mouse CD16/CD32 (Cat:101319), followed by anti-mouse antibodies, including anti-Ly6G-PE (Cat:127608), anti-CD11b-FITC (Cat:101205), and anti-F4/80-PE (Cat:123110). (A–C) The content of alveolar macrophages, neutrophils and monocytes in BALF at post-infection Day 0, Day 1, and Day 3 was examined by BD FACSCanto II flow cytometer and analyzed by FlowJo V10 software. (D) The number of total cells in BALF were determined using a Coulter Counter (Cellometer Auto 2000), and the number of alveolar macrophages, neutrophils and monocytes in BALF was calculated based on total cell number and their respective proportions. Note: The total cells were mixed cells from alveolar lavage fluid of seven, six and five mice in the day 0, day 1, and day 3 post-infection groups, respectively. Three independent experiments were performed. p < 0.05, * infected vs. uninfected mice. Abbreviations: AM, alveolar macrophages; Neu, neutrophils; Mo, monocytes.

            These findings indicated that in both genotypes of mice treated with hydrocortisone acetate and infected with A. fumigatus, many neutrophils and some monocytes were recruited into the lungs. However, deficiency in both pld1 and pld2 genes had little effect on the recruitment of neutrophils and monocytes into pulmonary alveoli.

            Effects of deficiency in both pld1 and pld2 genes on the release of inflammatory factors in BALF from immunosuppressed mice infected by A. fumigatus. Next, we examined the effects of deficiency in PLD on the release of inflammatory factors in BALF from immunosuppressed mice with A. fumigatus infection. During A. fumigatus infection, the concentrations of the chemokine MCP-1, and the pro-inflammatory cytokines TNF-α, IL-6, and Gro-αKC (IL-8) in BALF increased significantly in wild type and pld1-/-pld2-/- mice; the increase was clearer in pld1-/-pld2-/- mice at day 1 and day 3 after A. fumigatus infection (Fig 4AD). The concentration of IL-10, an anti-inflammatory cytokine, was higher in pld1-/-pld2-/- mice than wild type mice at day 1 and remained high through day 5 (Fig 4E). Unexpectedly, the concentration of IL-12p40 increased dramatically on day 3 and day 5 in wild type mice but not pld1-/-pld2-/- mice (Fig 4F).

            FIGURE 4 |

            In immunosuppressed mice, deficiency in both pld1 and pld2 genes significantly suppresses the production of IL-12p40 and promotes the production of IL-10 in BALF during A. fumigatus infection.

            Mice with immunosuppression were inoculated intranasally with 5×106 CFU conidia and anesthetized by intraperitoneal administration of 25% (v/v) ethyl carbamate. BALF was collected at post-infection day 0, day 1, day 3, and day 5. Each group included six or seven mice. The concentrations of inflammatory cytokines MCP-1 (A), TNF-α (B), IL-6 (C), Gro-αKC (D), IL-10 (E) and IL-12p40 (F) in BALF were detected with multiplex immunoassays according to the manufacturer’s instructions (eBioscience). Statistically significant differences were determined with one-way ANOVA followed by Tukey post-hoc test (vs day 0, ***p < 0.01, **p < 0.01 and *p < 0.05). The significance of differences was determined with unpaired Student’s t-tests with a 95% confidence interval (vs wild type, # p < 0.05, ## p < 0.01, and ### p < 0.001) in GraphPad Prism 8.0.2 software.

            These findings suggested that A. fumigatus infection significantly increased the content of the inflammatory factors MCP-1, TNF-α, IL-6, Gro-αKC, and IL-10 in the alveolar lavage fluid of mice under hydrocortisone acetate induced immunosuppression. Deficiency in both pld1 and pld2 genes in mice promoted the release of IL-10 and suppressed the release of IL-12p40 during A. fumigatus infection.

            DISCUSSION

            Despite being discovered several decades ago, the role of PLD continues to be more precisely elucidated. Most studies have investigated the functions of PLD in cancer, aging-associated diseases, thrombosis, and multiple sclerosis, thus indicating its potential as a target for therapeutic intervention [13]. Most mammalian PLD research has focused on PLD1 and PLD2. Other potential mammalian isoforms have been reported, including PLD3 (also called Sam-9 or HUK4), PLD4, PLD5, and PLD6 (also called mitochondrial PLD, MitoPLD, Zucchini, or Zuc). To date, no studies have been reported whether PLDs in mammalian cells are involved in host antifungal infections. This study presents the first in vivo data on PLD function against Aspergillus infection in mice.

            In immunocompetent and immunosuppressive mice, no significant difference was observed in the number of alveolar macrophages in BALF between wild type and pld1-/-pld2-/- mice before and after A. fumigatus infection, thus indicating that the absence of PLD did not alter the number of macrophages in the alveoli. However, in our previous study, the fungal burden in lung tissue and BALF was significantly higher in pld1-/-pld2-/- mice than wild type mice at 6 h post-administration in immunocompetent mice, and the phagocytosis and the fungicidal ability of isolated bone marrow-derived macrophages were significantly lower in pld1-/-pld2-/- mice than wild type mice [24]. In addition, before A. fumigatus infection, the content and number of alveolar macrophages in BALF from immunosuppressed mice were significantly lower than those in mice with normal immune function, but no such changes were observed in neutrophils and monocytes. After A. fumigatus infection, the content and number of alveolar macrophages in BALF sharply decreased, whereas the content and number of neutrophils and monocytes sharply increased in immunosuppressed mice. Moreover, other studies have demonstrated that corticosteroids impair the killing of A. fumigatus conidia by macrophages [18,27,28]. After phagocytosis by macrophages, another important fungal pathogen, Candida albicans, transitions from the yeast to the hyphal stage, and causes macrophage death and fungal escape [29]. Hence, macrophage damage by hydrocortisone acetate and phagocytosis-associated death might possibly explain the rapid and irreversible decrease in alveolar macrophages in BALF in hydrocortisone acetate-immunosuppressed mice during A. fumigatus infection. Moreover, the damage might have contributed to the increased susceptibility observed in the pld1-/-pld2-/- mice in response to A. fumigatus infection.

            Neutrophils play critical roles in host defense against invading microorganisms. Previous studies have shown that PLD activation is an early event in β2 integrin-mediated phagocytosis in neutrophils [14], and both PLD1 and PLD2 coordinately regulate cell migration [17]. PLD enzyme activity in neutrophils and leukocytes is associated with antimicrobial functions, such as phagocytosis, chemotaxis, and membrane ruffling [18]. pld1-/- and pld2-/- neutrophils exhibit impaired migration in vitro, but only pld1-/- neutrophils have impaired tissue extravasation [15]. In this study, in both immunocompetent and immunosuppressive states, the number of neutrophils in BALF increased significantly in response to A. fumigatus infection, particularly in mice with immunosuppression induced by hydrocortisone acetate, and the content and number of neutrophils in BALF sharply increased after A. fumigatus infection. However, no significant difference was observed in the number of neutrophils of BALF between wild type mice and pld1-/-pld2-/- mice. Loss of PLD probably did not affect the recruitment of neutrophils to the alveoli in mice during A. fumigatus infection, but instead directly modulated the phagocytosis and fungicidal activity of neutrophils toward A. fumigatus. In mouse models of sepsis, PLD2 in neutrophils is essential for the pathogenesis of experimental sepsis. Bactericidal activity is significantly elevated in pld2-/- mice, and is mediated by increased neutrophil extracellular trap formation and citrullination of histone 3 through peptidylarginine deiminase activation [25]. The exact mechanism of regulation of PLD on the function of neutrophils against A. fumigatus in vivo requires further exploration. Isolating primary neutrophils from pld1-/-pld2-/- mice for further investigation of aspects such as phagocytosis, chemotaxis, and extracellular traps should prove interesting.

            In immunocompetent states, after inhalation of A. fumigatus conidia, the content of inflammatory cytokines IL-6, Gro-αKC, and TNF-α in BALF from pld1-/-pld2-/- mice is significantly higher than that in wild type mice, particularly at 6 h after administration [24]. Similarly, we observed that, in immunosuppressive states, the content of these inflammatory cytokines in BALF from pld1-/-pld2-/- mice was also slightly higher than that in wild type mice at day 1 and/or day 3 post-infection, although no statistical difference was observed. This phenomenon might be explained by deficiency in PLD inducing greater secretion of inflammatory cytokines, which in turn recruit immune cells to combat A. fumigatus infection. In contrast, an excessive inflammatory response is also unfavorable to the host and may cause acute inflammatory injury [30,31]. In addition, the cytokine response was likely to be proportional to the higher fungal burden observed in the pld1-/-pld2-/- mice than wild type mice.

            IL-12—consisting of two subunits, IL-12p40 and IL-12p35—is an important immunoregulatory cytokine with multiple biological functions. It is produced mainly by phagocytes (monocytes/macrophages and neutrophils) and dendritic cells, in response to pathogens (bacteria, viruses, intracellular parasites, and fungi) [32,33]. IL-12 protects mice against A. fumigatus and C. albicans infection. Moreover, recombinant human interleukin-12 (IL-12) enhances the ability of human monocytes to elicit an oxidative burst and damage the hyphae of A. fumigatus [34]. Another two members of IL-12 family, IL-23 and IL-27, are involved in the response to A. fumigatus infection [32,35]. However, whether loss or abnormal function of host PLD affects the production of IL-12 during A. fumigatus infection is unknown. In this study, the deletion of two major PLDs, PLD1 and PLD2, significantly inhibited the production of IL-12p40 in mice in response to A. fumigatus infection. However, the exact mechanism of this inhibition by PLD on IL-12p40 release during A. fumigatus infection must be further studied.

            IL-10, an immunosuppressive and anti-inflammatory cytokine, is essential in maintaining peripheral immunological tolerance and limiting inflammation-associated tissue damage. Blocking IL-10 signaling enhances fungal clearance during cryptococcal infection in the lungs, and IL-10 blockade might be a potential therapy for the treatment of fungal lung infections [36,37]. IL-10 is deleterious during systemic aspergillosis infection, thereby increasing host susceptibility to lethal infection [38]. During A. fumigatus infection, the concentration of IL-10 in the BALF from pld1-/-pld2-/- mice was significantly higher than that from wild type mice; this aspect was likely to be another essential factor in the higher fungal burden and earlier death of pld1-/-pld2-/- mice than wild type mice.

            In summary, this was the first study using pld1-/-pld2-/- mice to investigate the function of PLD against fungal infection in either immunocompetent or immunosuppressed mice. The findings indicated that deficiency in both the pld1 and pld2 genes in mice in vivo makes immunosuppressed mice more susceptible to A. fumigatus infection and significantly affects the release of inflammatory factors during A. fumigatus infection.

            CONFLICTS OF INTEREST

            The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

            REFERENCES

            1. McCormick A, Loeffler J, Ebel F. Aspergillus fumigatus: contours of an opportunistic human pathogen. Cell Microbiol. 2010. Vol. 12(11):1535–1543

            2. Tischler BY, Hohl TM. Menacing mold: recent advances in Aspergillus pathogenesis and host defense. J Mol Biol. 2019. Vol. 431(21):4229–4246

            3. Desoubeaux G, Cray C. Rodent models of invasive aspergillosis due to Aspergillus fumigatus: still a long path toward standardization. Front Microbiol. 2017. Vol. 8:841

            4. Bitar D, Lortholary O, Le Strat Y, Nicolau J, Coignard B, Tattevin P, et al.. Population-based analysis of invasive fungal infections, France, 2001-2010. Emerg Infect Dis. 2014. Vol. 20(7):1149–1155

            5. Heinekamp T, Schmidt H, Lapp K, Pähtz V, Shopova I, Köster-Eiserfunke N, et al.. Interference of Aspergillus fumigatus with the immune response. Semin Immunopathol. 2015. Vol. 37(2):141–152

            6. Balloy V, Huerre M, Latgé JP, Chignard M. Differences in patterns of infection and inflammation for corticosteroid treatment and chemotherapy in experimental invasive pulmonary aspergillosis. Infect Immun. 2005. Vol. 73(1):494–503

            7. Ramirez-Ortiz ZG, Means TK. The role of dendritic cells in the innate recognition of pathogenic fungi (A. fumigatus, C. neoformans and C. albicans). Virulence. 2012. Vol. 3(7):635–646

            8. Gersuk GM, Underhill DM, Zhu L, Marr KA. Dectin-1 and TLRs permit macrophages to distinguish between different Aspergillus fumigatus cellular states. J Immunol. 2006. Vol. 176(6):3717–3724

            9. Soehnlein O, Lindbom L, Weber C. Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood. 2009. Vol. 114(21):4613–4623

            10. Medzhitov R. Origin and physiological roles of inflammation. Nature. 2008. Vol. 454(7203):428–435

            11. Sakai H, Sakane F. Recent progress on type II diacylglycerol kinases: the physiological functions of diacylglycerol kinase delta, eta and kappa and their involvement in disease. J Biochem. 2012. Vol. 152(5):397–406

            12. Frohman MA. The phospholipase D superfamily as therapeutic targets. Trends Pharmacol Sci. 2015. Vol. 36(3):137–144

            13. McDermott MI, Wang Y, Wakelam MJO, Bankaitis VA. Mammalian phospholipase D: function, and therapeutics. Prog Lipid Res. 2020. Vol. 78:101018

            14. Serrander L, Fallman M, Stendahl O. Activation of phospholipase D is an early event in integrin-mediated signalling leading to phagocytosis in human neutrophils. Inflammation. 1996. Vol. 20(4):439–450

            15. Ali WH, Chen Q, Delgiorno KE, Su W, Hall JC, Hongu T, et al.. Deficiencies of the lipid-signaling enzymes phospholipase D1 and D2 alter cytoskeletal organization, macrophage phagocytosis, and cytokine-stimulated neutrophil recruitment. PLoS One. 2013. Vol. 8(1):e55325

            16. Iyer SS, Barton JA, Bourgoin S, Kusner DJ. Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis. J Immunol. 2004. Vol. 173(4):2615–2623

            17. Knapek K, Frondorf K, Post J, Short S, Cox D, Gomez-Cambronero J. The molecular basis of phospholipase D2-induced chemotaxis: elucidation of differential pathways in macrophages and fibroblasts. Mol Cell Biol. 2010. Vol. 30(18):4492–4506

            18. Brown HA, Thomas PG, Lindsley CW. Targeting phospholipase D in cancer, infection and neurodegenerative disorders. Nat Rev Drug Discov. 2017. Vol. 16(5):351–367

            19. Jia X, Chen F, Pan W, Yu R, Tian S, Han G, et al.. Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation. Microbes Infect. 2014. Vol. 16(6):491–501

            20. Han X, Yu R, Zhen D, Tao S, Schmidt M, Han L, et al.. beta-1,3-Glucan-induced host phospholipase D activation is involved in Aspergillus fumigatus internalization into type II human pneumocyte A549 cells. PLoS One. 2011. Vol. 6(7):e21468

            21. Sugui JA, Pardo J, Chang YC, Zarember KA, Nardone G, Galvez EM, et al.. Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone. Eukaryot Cell. 2007. Vol. 6(9):1562–1569

            22. Zhang C, Chen F, Liu X, Han X, Hu Y, Su X, et al.. Gliotoxin induces cofilin phosphorylation to promote actin cytoskeleton dynamics and internalization of Aspergillus fumigatus into type II human pneumocyte cells. Front Microbiol. 2019. Vol. 10:1345

            23. Shevchenko MA, Bogorodskiy AO, Troyanova NI, Servuli EA, Bolkhovitina EL, Büldt G, et al.. Aspergillus fumigatus infection-induced neutrophil recruitment and location in the conducting airway of immunocompetent, neutropenic, and immunosuppressed mice. J Immunol Res. 2018. Vol. 2018:5379085

            24. Liu Xiaoyu Zhang Changjian, Yingsong H, Fangyan C, Li H. Loss of phospholipase D in mice against Aspergillus fumigatus infection. Chin J Mycol. 2018. Vol. 13(3):129–133

            25. Lee SK, Kim SD, Kook M, Lee HY, Ghim J, Choi Y, et al.. Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2. J Exp Med. 2015. Vol. 212(9):1381–1390

            26. Kantonen S, Hatton N, Mahankali M, Henkels KM, Park H, Cox D, et al.. A novel phospholipase D2-Grb2-WASp heterotrimer regulates leukocyte phagocytosis in a two-step mechanism. Mol Cell Biol. 2011. Vol. 31(22):4524–4537

            27. Schaffner A, Douglas H, Braude A. Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes. J Clin Invest. 1982. Vol. 69(3):617–631

            28. Merkow L, Pardo M, Epstein SM, Verney E, Sidransky H. Lysosomal stability during phagocytosis of Aspergillus flavus spores by alveolar macrophages of cortisone-treated mice. Science. 1968. Vol. 160(3823):79–81

            29. Uwamahoro N, Verma-Gaur J, Shen HH, Qu Y, Lewis R, Lu J, et al.. The pathogen Candida albicans hijacks pyroptosis for escape from macrophages. mBio. 2014. Vol. 5(2):e00003–14

            30. Yu B, Li C, Gu L, Zhang L, Wang Q, Zhang Y, et al.. Eugenol protects against Aspergillus fumigatus keratitis by inhibiting inflammatory response and reducing fungal load. Eur J Pharmacol. 2022. Vol. 924:174955

            31. Van Woensel J, Leers MP, Mostard RL. Invasive pulmonary aspergillosis with marked eosinophilia as a unique presentation of chronic granulomatous disease. Eur J Case Rep Intern Med. 2022. Vol. 9(7):003423

            32. Kowalewska B, Zorena K, Szmigiero-Kawko M, Wa˛z˙ P, Mys´liwiec M. High interleukin-12 levels may prevent an increase in the amount of fungi in the gastrointestinal tract during the first years of diabetes mellitus type 1. Dis Markers. 2016. Vol. 2016:4685976

            33. Cenci E, Mencacci A, Fè d’Ostiani C, Del Sero G, Mosci P, Montagnoli C, et al.. Cytokine- and T helper-dependent lung mucosal immunity in mice with invasive pulmonary aspergillosis. J Infect Dis. 1998. Vol. 178(6):1750–1760

            34. Roilides E, Tsaparidou S, Kadiltsoglou I, Sein T, Walsh TJ. Interleukin-12 enhances antifungal activity of human mononuclear phagocytes against Aspergillus fumigatus: implications for a gamma interferon-independent pathway. Infect Immun. 1999. Vol. 67(6):3047–3050

            35. Gafa V, Lande R, Gagliardi MC, Severa M, Giacomini E, Remoli ME, et al.. Human dendritic cells following Aspergillus fumigatus infection express the CCR7 receptor and a differential pattern of interleukin-12 (IL-12), IL-23, and IL-27 cytokines, which lead to a Th1 response. Infect Immun. 2006. Vol. 74(3):1480–1489

            36. Teitz-Tennenbaum S, Viglianti SP, Roussey JA, Levitz SM, Olszewski MA, Osterholzer JJ. Autocrine IL-10 signaling promotes dendritic cell type-2 activation and persistence of murine cryptococcal lung infection. J Immunol. 2018. Vol. 201(7):2004–2015

            37. Murdock BJ, Teitz-Tennenbaum S, Chen GH, Dils AJ, Malachowski AN, Curtis JL, et al.. Early or late IL-10 blockade enhances Th1 and Th17 effector responses and promotes fungal clearance in mice with cryptococcal lung infection. J Immunol. 2014. Vol. 193(8):4107–4116

            38. Clemons KV, Grunig G, Sobel RA, Mirels LF, Rennick DM, Stevens DA. Role of IL-10 in invasive aspergillosis: increased resistance of IL-10 gene knockout mice to lethal systemic aspergillosis. Clin Exp Immunol. 2000. Vol. 122(2):186–191

            Author and article information

            Journal
            Zoonoses
            Zoonoses
            Zoonoses
            Compuscript (Shannon, Ireland )
            2737-7466
            2737-7474
            11 May 2023
            : 3
            : 1
            : e977
            Affiliations
            [1 ]Department for Disinfection and Infection Control, Chinese PLA Center for Disease Control and Prevention, Beijing, China
            [2 ]The 920th Hospital of Joint Logistics Support Force, PLA, Kunming, China
            Author notes
            *Corresponding author: E-mail: hanlicdc@ 123456163.com (LH)

            #These authors contributed equally to this article.

            Article
            10.15212/ZOONOSES-2022-0044
            f2cded13-bedb-4295-b401-83ad8999ca4d
            Copyright © 2023 The Authors.

            This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY) 4.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

            History
            : 01 November 2022
            : 25 February 2023
            : 11 April 2023
            Page count
            Figures: 4, References: 38, Pages: 10
            Funding
            Funded by: National Natural Science Foundation of China
            Award ID: 81971914
            Funded by: National Natural Science Foundation of China
            Award ID: 81401305
            Funded by: National Natural Science Foundation of China
            Award ID: 81571542
            We thank Prof. Dr. Bernhard Nieswandt for kindly providing pld1 and pld2 gene double-deficient mice ( pld1 -/-/pld2 -/- ), and Dr. K. J. Kwon-Chung for kindly providing the A. fumigatus wild type strain B5233. No potential conflicts of interest are disclosed. This work was supported by the National Natural Science Foundation of China (Grants No. 81971914, No. 81401305, and No. 81571542). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.
            Categories
            Original Article

            Parasitology,Animal science & Zoology,Molecular biology,Public health,Microbiology & Virology,Infectious disease & Microbiology
            neutrophils,phospholipase D,inflammatory cytokines, Aspergillus fumigatus ,macrophages

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