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. 2023 Jan 5;91(1):e00378-22. doi: 10.1128/iai.00378-22

The Protective Role of Interleukin 17A in Acinetobacter baumannii Pneumonia Is Associated with Candida albicans in the Airway

Jiaxin Tang a, Jiamin Li a, Jinqing Pan a, Xiaoyuan Shen a, Xiangsheng Ye a, Jiamin Zhou a, Ni Wang a, Liang Xie a, Beth Burgwyn Fuchs b, Michail S Lionakis c,✉, Eleftherios Mylonakis b,✉, Xiaojiang Tan a,✉
Editor: Mairi C Noverrd
PMCID: PMC9872622  PMID: 36602381

ABSTRACT

Recent studies have found that the coexistence of fungi and bacteria in the airway may increase the risk of infection, contribute to the development of pneumonia, and increase the severity of disease. Interleukin 17A (IL-17A) plays important roles in host resistance to bacterial and fungal infections. The objective of this study was to determine the effects of IL-17A on Acinetobacter baumannii-infected rats with a previous Candida albicans airway inoculation. The incidence of A. baumannii pneumonia was higher in rats with C. albicans in the airway than in noninoculated rats, and it decreased when amphotericin B was used to clear C. albicans, which influenced IL-17A levels. IL-17A had a protective effect in A. baumannii pneumonia associated with C. albicans in the airway. Compared with A. baumannii-infected rats with C. albicans in the airway that did not receive IL-17A, recombinant IL-17A (rIL-17A) supplementation decreased the incidence of A. baumannii pneumonia (10/15 versus 5/17; P = 0.013) and the proportion of neutrophils in the lung (84 ± 3.5 versus 74 ± 4.3%; P = 0.033), reduced tissue destruction and inflammation, and decreased levels of myeloperoxidase (MPO) (1.267 ± 0.15 versus 0.233 ± 0.06 U/g; P = 0.0004), reactive oxygen species (ROS) (132,333 ± 7,505 versus 64,667 ± 10,115 AU; P = 0.0007) and lactate dehydrogenase (LDH) (2.736 ± 0.05 versus 2.1816 ± 0.29 U/g; P = 0.0313). In vitro experiments revealed that IL-17A had no significant effect on the direct migration ability and bactericidal capability of neutrophils. However, IL-17A restrained lysis cell death and increased apoptosis of neutrophils (2.9 ± 1.14 versus 7 ± 0.5%; P = 0.0048). Taken together, our results suggest that C. albicans can depress IL-17A levels, which when supplemented may have a regulatory function that limits the accumulation of neutrophils in inflammatory areas, providing inflammatory response homeostasis.

KEYWORDS: Candida albicans, Acinetobacter baumannii, interleukin 17A, neutrophils, apoptosis

INTRODUCTION

Acinetobacter baumannii infections have spread rapidly in hospitals around the world (1), with intensive care units (ICU) leading the way, experiencing high mortality rates of up to 60% (2). Bacterial infections can become more challenging when there is a secondary fungal infection. Candida albicans is part of the normal human flora and colonizes the airways of more than half of critically ill patients (3). In our previous study of ICU patients, we found that patients with C. albicans airway colonization had a higher incidence of A. baumannii pneumonia than noncolonized patients and that C. albicans airway colonization was an independent risk factor (4). We used a rat model to confirm that C. albicans in the airway promoted and aggravated A. baumannii pneumonia (5). Interestingly, the concentration of interleukin 17 (IL-17) in the lungs of C. albicans in the airway group was lower than that in the noninoculated group. Moreover, the incidence of A. baumannii pneumonia was lower and IL-17 levels were elevated in the airways of rats with C. albicans after receipt of antifungal treatment (5). This suggests that IL-17 levels may impact the incidence of A. baumannii pneumonia in this setting.

The IL-17 family, discovered in 1993, consists of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F (6). IL-17, commonly known as IL-17A, plays an important role in the immune defense against pathogen infection (7), providing protection against extracellular microbial attack and the progression of bacterial pneumonia (8–10), oral candidiasis (11, 12), and inflammatory bowel disease (13, 14).

Examples of IL-17 protective roles were demonstrated in a mouse Pseudomonas aeruginosa infection model that lacked γδ T cells and exhibited markedly reduced IL-17 and increased bacterial loads (15). IL-17 also demonstrated a potential protective role in Pneumocystis pneumonia by reducing lung injury and increasing fungal clearance (10). Ethanol-treated mice infected with Klebsiella pneumoniae had decreased IL-17 release, resulting in reduced neutrophil recruitment and increased mortality (16). In addition, studies have reported that IL-17A can regulate neutrophil homeostasis, promote neutrophil apoptosis, and relieve inflammatory responses (17, 18).

Our study investigated and compared the role of IL-17A in host defense against A. baumannii associated with C. albicans in the airway. The results suggest that decreased levels of IL-17A are associated with increased incidence of A. baumannii pneumonia. Compared with A. baumannii-infected rats with C. albicans in the airway that did not receive IL-17A, recombinant IL-17A (rIL-17A) supplementation decreased the incidence of A. baumannii pneumonia and reduced neutrophil accumulation and associated excessive inflammatory response and tissue destruction. In vitro experiments revealed that IL-17A had no significant effect on the direct migration ability and bactericidal capability of neutrophils. However, IL-17A supplementation reduced neutrophil accumulation and excessive inflammation associated with increased neutrophil apoptosis. These findings suggest that IL-17A had a protective effect in A. baumannii pneumonia associated with C. albicans in the airway.

RESULTS

Fungal airway inoculation facilitates the development of subsequent A. baumannii pneumonia.

Using a previously described model (5, 19), C. albicans was inoculated into the airways of Wistar rats. As shown in Fig. 1A, the 14-day survival of the C. albicans-inoculated rats (Ca rats) was 100%, and there was no difference in activity, fecal shape, and body weight compared with the control rats (normal saline) (Fig. 1A and B). There was no histopathologic evidence of pneumonia in C. albicans-inoculated rats (Fig. 1C to E), and C. albicans was not found in the liver or kidney.

FIG 1.

FIG 1

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The presence of C. albicans in the rat airway facilitates development of subsequent A. baumannii pneumonia. (A to E) A total of 3 × 106 CFU of C. albicans was transglottally inoculated into rats. At different time points, the rats were sacrificed, and their lungs were homogenized. (A) C. albicans CFU counts at 4 h, 24 h, 3 days, 7 days, and 14 days after inoculation. Four animals were sacrificed at each time point. (B) Animal weight was compared with that of noninoculated rats (control) at each time point. (C to E) Twenty-four hours after lungs were inoculated with C. albicans or saline, both sets of lungs were normal in macroscopic appearance, axial CT imaging, and microscopic appearance (hematoxylin-eosin stain; magnification, ×400). (F to J) For additional assessment, rats were divided into those with previous C. albicans airway inoculation (n = 15) and saline controls (n = 15). A suspension of 108 CFU of A. baumannii ATCC 19606 was transglottally inoculated into each animal on the second day of the experiment. (F) Median A. baumannii CFU counts per lung (log transformed). The black line indicates a bacterial burden of 104 CFU per lung. (G) Normal macroscopic appearance of lungs from rats that were not colonized with C. albicans (Ab group) and did not develop pneumonia after transglottal inoculation of A. baumannii. Marked consolidation in the lung (red arrow) of a rat colonized with C. albicans that developed pneumonia after instillation of the same A. baumannii inoculum (AWC group). (H) Lung axial CT images showing obvious consolidation in rats with A. baumannii infection after previous C. albicans inoculation (AWC rats). There are multiple patchy or patchy infiltrating shadows, and a bronchial air sign is seen within the lesion (red arrow). (I) Microscopic appearance of an A. baumannii-instilled lung by light microscopy (hematoxylin-eosin stain; magnification, ×200). Heavier infiltration of inflammatory cells and alveolar damage is seen in AWC rats. (J) Median C. albicans CFU counts per lung (log transformed). The error bars indicate standard deviations (SD). Data were analyzed by the Mann-Whitney test. ns, P > 0.05; *, P < 0.05.

According to the study by Roux et al. (19), which also evaluated the effect of C. albicans in the airway on different types of bacterial pneumonia, pneumonia was defined as macroscopic and/or microscopic lung inflammation with a bacterial burden of >104 CFU per lung. Twenty-four hours after lung inoculation of A. baumannii, 10 of 15 rats with C. albicans in the airway (AWC rats) developed A. baumannii pneumonia, as opposed to 4/15 in the control rats (Ab rats) (P = 0.028) (Fig. 1F). Rats with C. albicans in the airway prior to A. baumannii had higher lung bacterial CFU counts (P = 0.015) than controls (Fig. 1F). Consolidation was evident in the lung of a C. albicans-inoculated rat (Fig. 1G to I), and there was no statistical difference in numbers of C. albicans CFU with and without A. baumannii infection (P = 0.273) (Fig. 1J).

Previous fungal inoculation into the airway downregulated host IL-17 production.

To further investigate the effects of C. albicans inoculation prior to A. baumannii infection on rat host immune response, we measured the levels of lung granulocyte colony-stimulating factor (G-CSF), MIP-1α, IL-1β, IL-2, IL-6, IL-17A, and other cytokines in different rat groups. Levels of gamma interferon (IFN-γ) (Fig. 2A), IL-2 (Fig. 2D), and IL-17A (Fig. 2I) were higher in Ca rats than in controls. AWC rats had significantly higher IFN-γ (464.6 ± 121.4 versus 232.2 ± 31.4 pg/mL; P = 0.039) (Fig. 2A) but lower IL-17A concentrations (34.26 ± 3.34 versus 42.12 ± 3.04 pg/mL; P = 0.013) than Ab rats (Fig. 2I). There was no difference in the levels of other cytokines.

FIG 2.

FIG 2

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C. albicans in the airway alters the host immune response to A. baumannii infection. Levels of IFN-γ, TNF-α, IL-1β, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17A, G-CSF, MIP-1α, and MIP-2 in the absence of A. baumannii or 24 h after A. baumannii inoculation in C. albicans-inoculated rats versus controls (normal saline). Data were analyzed by Student’s t test. Data are means and SD. *, P < 0.05.

Antifungal treatment reduced C. albicans load and resulted in elevated IL-17A.

In an additional series of experiments, we used amphotericin B to decrease the CFU of C. albicans and evaluated the incidence of A. baumannii pneumonia. Systemic amphotericin B (1 mg/kg of body weight/day) was administered for 3 consecutive days, beginning on the day of C. albicans lung inoculation. The results showed a significant reduction in fungal burden in the amphotericin B treatment group (10.5 × 104 versus 0.38 × 104; P = 0.0012) (Fig. 3A), and the incidence of A. baumannii pneumonia was reduced (10/15 versus 5/18; P = 0.0131) (Fig. 3B). Notably, IL-17A levels increased (35.68 ± 3.99 versus 41.32 ± 7.46 pg/mL; P = 0.0132) (Fig. 3C).

FIG 3.

FIG 3

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Effect of antifungal therapy. (A) Fungal load in lung homogenates was assessed by plating the material and enumerating the CFU. (B) Lung homogenates were also plated to enumerate bacterial load. (C) The level of IL-17A in rat lungs was measured in the two groups. Fungal and bacterial loads were analyzed by the Mann-Whitney test, and IL-17A levels were analyzed by Student’s t test. Data are means and SD. *, P < 0.05; **, P < 0.01.

IL-17A plays a protective role in A. baumannii pneumonia when C. albicans is also in the airway.

It appears that a higher incidence of A. baumannii pneumonia was associated with lower concentrations of IL-17A in lung homogenates. To define the role of IL-17A in host defense against A. baumannii infection in this setting, A. baumannii-infected rats with C. albicans in the airway received recombinant IL-17A supplementation (AWC+IL-17A). IL-17A was elevated in AWC+IL-17A rats (Fig. 4A). Five of 17 rats in the AWC+IL-17A group developed A. baumannii pneumonia, as opposed to 10 of 15 in the AWC group (P = 0.013), and bacterial CFU (P = 0.011) (Fig. 4B) were significantly decreased. However, there was no statistical difference in C. albicans CFU with or without recombinant IL-17A supplementation (P = 0.712) (Fig. 4C). Macroscopic lung examination (Fig. 4D), lung computed tomography (CT) imaging (Fig. 4E), and lung histology (Fig. 4F) showed a significant reduction in lung infection and inflammation upon recombinant IL-17A supplementation.

FIG 4.

FIG 4

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IL-17A has a protective effect against pulmonary infection with A. baumannii by reducing lung inflammation and enhancing bacterial clearance. Rats were divided into AWC (n = 15) and AWC+IL-17A (n = 17) groups. rIL-17A (2 mg/kg) was injected intraperitoneally 2 h before and after transglottal instillation of A. baumannii. (A) Levels of IL-17A in rat lungs. (B) Median A. baumannii CFU counts per lung (log transformed). The black line indicates a bacterial burden of 104 CFU per lung. (C) Median C. albicans CFU counts per lung (log transformed). (D) Macroscopic appearance of the rat lungs. The red arrows point to multiple large inflammatory changes evident in the lung. (E) Lung axial CT images show obvious consolidation in rats with A. baumannii infection after previous C. albicans inoculation (AWC rats). There are multiple patchy or patchy infiltrating shadows, and a bronchial air sign is seen within the lesion (red arrow). (F) Microscopic appearance of lung by light microscopy (hematoxylin-eosin stain; magnification, ×200). Data were analyzed by the Mann-Whitney test and Student’s t test. Data are means and SD. ns, P > 0.05; *, P < 0.05; **, P < 0.01.

IL-17A alters the host immune and inflammatory response to A. baumannii-infected rats with C. albicans in the airway.

To further investigate the effects of IL-17A on the host immune response in A. baumannii-infected rats with previous C. albicans inoculation, we measured the total number of cells and the proportions of neutrophils and macrophages in bronchoalveolar lavage fluid (BALF). Compared with AWC rats, the total cell counts (213 × 105 ± 21.9 × 105 versus 96.3 × 105 ± 10.8 × 105; P = 0.0012) were decreased after rIL-17A supplementation (Fig. 5A), particularly the proportion of neutrophils (84 ± 3.5% versus 74 ± 4.3%; P = 0.033) (Fig. 5B) and the overall number of neutrophils (178.9  × 105± 14.1 × 105 versus 71.6 × 105 ± 11.8 × 105; P = 0.0005) (Fig. 5C). In contrast, the proportion of macrophages increased (7.4% ± 1.8% versus 14.5% ± 2%; P = 0.0103) in BALF (Fig. 5D). However, there was no difference in the overall number of macrophages between the two groups (16.01 × 105 ± 5.3 × 105 versus 14.01 × 105 ± 2.9 × 105; P = 0.601) (Fig. 5E). Thus, the macrophage proportion difference is likely an effect of neutrophil depletion.

FIG 5.

FIG 5

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IL-17A reduces the number of neutrophils and neutrophil-related pulmonary inflammatory responses in A. baumannii-infected rats with C. albicans in the airway. (A) The total number of BALF cells was determined with a hemocytometer. (B to E) Differential counts of macrophages and neutrophils were determined by flow cytometry. (F) MPO activities in lung homogenates of rats were measured. (G) ROS was measured in cells from BALF. (H) LDH was measured in the supernatant of pulmonary tissue homogenates. (I to K) Levels of TNF-α, IL-1β, and IL-6 were measured in the rat lungs. Data are means and SD. Data were analyzed by Student’s t test. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Neutrophils are the most abundant phagocytes and play an important role in the host defense against invading pathogens (20, 21). Neutrophils accumulate in infected tissues to help the body fight off infections, but excessive neutrophil accumulation can damage tissues, leading to excessive inflammation and immunopathology (22, 23). To further determine the lung inflammatory responses, we examined secreted myeloperoxidase (MPO), reactive oxygen species (ROS), lactate dehydrogenase (LDH), and the proinflammatory cytokines TNF-α, IL-6, and IL-1β. The activity of MPO, mostly expressed in neutrophils, was significantly increased in lung homogenates of A. baumannii-infected rats with C. albicans in the airway, and it decreased upon supplement with rIL-17A (1.267 ± 0.15 versus 0.233 ± 0.06 U/g; P = 0.0004) (Fig. 5F). Similarly, ROS production (64,667 ± 10,115 versus 132,333 ± 7,505 AU; P = 0.0007) (Fig. 5G) and LDH release (2.1816 ± 0.29 versus 2.736 ± 0.05 U/g; P = 0.0313) (Fig. 5H) decreased significantly in the rIL-17A group. In contrast, TNF-α, IL-6, and IL-1β were not altered (Fig. 5I to K). Neutrophils are a major contributor to excessive inflammatory responses (24) and were observed to increased significantly in A. baumannii-infected rats with C. albicans in the airway. After IL-17A supplementation, neutrophils were significantly reduced in AWC rats, followed by a significant reduction in neutrophil-related pulmonary inflammatory response.

IL-17A has no direct effect on antibacterial ability and migration of neutrophils in response to A. baumannii.

IL-17A is mainly produced by activated CD4+ T cells, γδ T cells, and innate lymphoid cells (25, 26). During infection, IL-17A participates in the host’s innate immune response by recruiting granulocytes, promoting the release of inflammatory cytokines and enhancing the host’s ability to eliminate pathogens (11). Our in vivo results showed that although the number of neutrophils decreased, bacterial loads were also reduced after IL-17A supplementation. We reasoned that IL-17A may affect the ability of neutrophils to clear A. baumannii. Therefore, we investigated the phagocytic ability, bacterial killing ability, and migration of neutrophils against A. baumannii upon treatment with rIL-17A. Bacterial phagocytosis and killing did not demonstrate a difference in neutrophils after pretreatment with rIL-17A (Fig. 6A and B). In addition, IL-17A did not directly affect the migration of neutrophils (Fig. 6C).

FIG 6.

FIG 6

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Effects of IL-17A on neutrophil phagocytosis, bacterial killing and migration. (A to C) Neutrophils were isolated from the blood of WT rats. (A and B) Neutrophils were pretreated with or without 0.1 mg/mL of rIL-17A for 2 h and subsequently infected with A. baumannii at 1/10 MOI followed by gentamicin treatment 60 min after infection to remove extracellular bacteria. Live bacteria were then counted by plating onto LB agar supplemented with ampicillin (50 μg/mL) at 1 h (for phagocytosis) or 6 h (for bacterial killing) after infection with A. baumannii. (C) Cell suspension (100 μL; 2 × 105 cells) was added to the upper Transwell chamber, and 1,000 μL of a 1/10-MOI A. baumannii suspension with or without 0.1 mg/mL of rIL-17A was added to the lower chamber. The Transwell chambers were placed in an incubator with 5% CO2 and incubated at 37°C for 2 h. The number of neutrophils that migrated to the lower chamber was measured with a hemocytometer. Data are means and SD (n = 6). Data were analyzed by the Mann-Whitney test and Student’s t test. ns, not significant.

IL-17A reduces pyroptosis-associated lysis and increases apoptosis of neutrophils in A. baumannii-infected rats with C. albicans in the airway.

The above results showed that the IL-17A supplementation had no significant effect on the direct migration or bactericidal ability of neutrophils (Fig. 6). We further investigated the effect of IL-17A on neutrophil survival. When neutrophils complete their effector function during infection, they initiate spontaneous apoptosis and are cleared via phagocytosis by macrophages. If neutrophils undergo delayed apoptosis or initiate lysis, their excessive accumulation in tissues may cause tissue damage via enzymes, peptides, ROS, and other substances within neutrophils that are released (Fig. 5F to H).

Cell lysis is an inflammatory cell death caused primarily by two cellular mechanisms: pyroptosis and necroptosis (27). Pyroptosis depends on the cysteine protease caspase 1 and is associated with the release of large amounts of the proinflammatory cytokines IL-18 and IL-1β (28, 29). Necroptosis is another recently identified proinflammatory mode of lysis cell death, regulated by receptor-interacting serine-threonine kinases (RIPK1 to -3) and executed by mixed-lineage kinase domain-like protein (MLKL) (30). To detect activation of the neutrophils’ different death pathways, we harvested neutrophils in BALF by fluorescence-activated cell sorting (FACS), and protein and supernatant were extracted after cell lysis. We found that rIL-17A caused a decrease in neutrophilic caspase-1 activation and IL-18 production (70.39 versus 41.88 pg/mL; P = 0.0362) (Fig. 7A and B). Western blot experiments revealed no significant change of p-MLKL and RIPK3 expression in neutrophils between A. baumannii-infected rats with C. albicans in the airway and rats that received rIL-17A supplementation (Fig. 7C).

FIG 7.

FIG 7

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IL-17A affects neutrophil death from A. baumannii-infected rats with C. albicans in the airway. FACS was used to harvest neutrophils from BALF for subsequent experiments. (A) Immunoblot analysis of key molecules involved in pyroptosis. Neutrophils were analyzed for procaspase 1, cleaved caspase 1 (p-20), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). (B) ELISA of neutrophils for IL-18 secretion. (C) Immunoblot analysis of key molecules involved in necroptosis. Neutrophils were analyzed for RIPK3, p-MLKL, MLKL, and GAPDH. (D) Apoptosis of neutrophils was analyzed by flow cytometry using annexin V and 7-AAD. Representative FACS plots showing percentages of viable (live) and apoptotic or nonapoptotic dead neutrophils for each condition. (E) Fold change of caspase-3 activation in neutrophils of A. baumannii-infected rats with C. albicans in the airway was lower than that with rIL-17A supplementation. (F) Immunoblot analysis of key molecules involved in apoptosis. Neutrophils were analyzed for Bax, Bcl-2, and β-actin. Data are means and SD. Data were analyzed by Student’s t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Apoptosis is a nonlytic form of cell death, which differs from pyroptosis and necroptosis in that it is associated with the appearance of specific morphological features (31). Neutrophil apoptosis is least damaging to the surrounding tissue and is the main mechanism aimed at promoting inflammatory response resolution. We examined the kinetics of neutrophil apoptosis by flow cytometry, in which annexin V+/7-aminoactinomycin D (7-AAD)− cells represent early apoptotic cells and annexin V+/7-AAD+ cells represent late apoptotic/dead cells. We found that after rIL-17A supplementation in A. baumannii-infected rats with C. albicans in the airway, neutrophil apoptosis significantly increased (6.96 versus 2.9%; P = 0.0048) (Fig. 7D). Concordantly, caspase-3 activation (Fig. 7E) and Bax expression (Fig. 7F) increased and Bcl-2 expression (Fig. 7F) decreased in neutrophils upon rIL-17A supplementation. Collectively, our data show that IL-17A not only reduced pyroptosis-associated lysis cell death but also increased neutrophil apoptosis in the A. baumannii-infected rats with C. albicans in the airway.

DISCUSSION

Fungi and bacteria coexist in the body. Their cross interactions have coevolved, inclusive of infections, thus influencing each other’s virulence potential (19, 32–36). We found that previous C. albicans airway inoculation, which mimics colonization, facilitated the subsequent development of A. baumannii infection and decreased the levels of IL-17A, which is consistent with other animal studies (19) and clinical data (37, 38) for other pathogens. These findings support the notion that decreased IL-17A is associated with an increased incidence of A. baumannii pneumonia during previous airway inoculation with C. albicans. To confirm this, A. baumannii-infected rats with C. albicans in the airway received recombinant IL-17A supplementation. The incidence of pneumonia, lung bacterial burden, and results of macroscopic lung examination, histopathological analysis, and chest CT showed that IL-17A administration had a protective effect in A. baumannii-infected rats with C. albicans in the airway.

The IL-17 family of cytokines is a group of pleiotropic molecules that gained attention in recent years and can act as a double-edged sword (39); thus, IL-17A can drive inflammatory responses, but it also plays an important protective role in host defense after pathogen infection at mucocutaneous barriers, including lung infections (7, 34). The requirement for IL-17A for host defense varies with regard to extracellular fungi and bacterial pathogens, including Candida, Cryptococcus, Klebsiella, and Staphylococcus (35–37). IL-17A is a proinflammatory cytokine released by activated CD4+ (Th17 subsets) and CD8+ T lymphocytes and innate lymphoid cells, which can promote the production of chemokines and drive the migration of neutrophils and monocytes to infected or injured tissues (7). IL-17A can synergistically regulate IL-22 and participate in the maintenance of epithelial homeostasis and repair or regeneration of epithelial cells after inflammatory injury (40).

Innate immunity is the first line of defense against pathogens (41). Upon entry of microorganisms into the host, phagocytes such as macrophages and neutrophils are rapidly recruited to the site of infection and bind and ingest microorganisms. Our work found significant neutrophil increase in A. baumannii-infected rats with C. albicans in the airway and significantly increased levels of MPO and ROS, resulting in increased LDH and tissue damage. However, after rIL-17A supplementation, the number of neutrophils decreased, as did MPO, ROS, and LDH levels. In general, neutrophil depletion was found to sensitize mice to infection. An early mouse model of A. baumannii systemic infection used neutrophil-depleted animals to establish the infection (42). Although neutrophils are indispensable to fight lung infection, they can also be problematic (43–46): hyperactivity of neutrophil activation can cause severe tissue damage in the process of killing invading microorganisms as a result of releasing toxic substances, including protease, cationic peptides, cytokines, and ROS, intended to destroy invading pathogens. In extreme cases, damage caused by neutrophils and other innate immune mediators is a major source of morbidity and mortality.

In vitro cell experiments showed that IL-17A had no direct effect on the chemotaxis and the antibacterial ability of neutrophils. We further investigated the effect of IL-17A on neutrophil survival. Neutrophils have a half-life of 8 to 20 h, and most neutrophils undergo apoptosis within 24 h. When acute infection leads to an excessive inflammatory response, neutrophils exhibit delayed apoptosis, resulting in excessive accumulation of neutrophils in tissues, which aggravates tissue damage (46). In addition, excessive tissue destruction and inflammation are associated with neutrophil lysis, highlighting the importance of neutrophil apoptosis in the orderly resolution of infection (47–49).

A prior study of otitis media similarly showed that IL-17A contributes to the host immune response against Streptococcus pneumoniae by promoting neutrophil recruitment and apoptosis through the p38 MAPK signaling pathway (17). Moreover, Dragon et al. demonstrated that IL-17A may regulate neutrophil homeostasis and favor the resolution of inflammation in tissues by attenuating the delay in neutrophil apoptosis induced by inflammatory cytokines (18). In our study, A. baumannii-infected rats with C. albicans in the airway that received rIL-17A exhibited reduced neutrophil lysis via the pyroptosis pathway and increased neutrophil apoptosis relative to rats that did not receive rIL-17A. Thus, increased neutrophil apoptosis at the site of infection upon IL-17A administration appears to mitigate the bystander tissue destruction that occurs during lysis in this model.

In conclusion, we demonstrate that increased incidence of A. baumannii pneumonia in the setting of C. albicans in the airway is associated with decreased levels of IL-17A. IL-17A supplementation increases A. baumannii clearance and reduces neutrophil accumulation and excessive inflammatory responses. Moreover, IL-17A supplementation reduced pyroptosis-associated lysis death of neutrophils, increased apoptosis, and reduced excessive inflammatory responses and tissue injury. Collectively, our results suggest that IL-17A may have a regulatory function that limits the accumulation of neutrophils in inflammatory areas and promotes tissue homeostasis, which may provide a new target for the control of A. baumannii infection.

MATERIALS AND METHODS

Animals.

Male Wistar rats were purchased from the Experimental Animal Center of Southern Medical University, Guangzhou, China. All the experiments were conducted with 2.5- to 3-month-old male Wistar rats without pathogens, weighing 250 to 280 g. All experiments were approved by the Animal Experiment Ethics Committee of Nanfang Hospital, affiliated with Southern Medical University (NFYY-2020-0623). The study followed strict guidelines from The National Institutes of Health’s Guide for the Care and Use of Laboratory Animals (50).

Bacterial and fungal preparation.

A. baumannii (ATCC 19606) was purchased from Guangdong Microbial Species Preservation Center (Guangdong, China). Single colonies were inoculated into 10 mL of Luria-Bertani broth and grown overnight at 37°C with 200-rpm shaking for 16 h. Bacteria were washed and suspended with sterile phosphate-buffered saline (PBS) to a final concentration of 5 × 108 CFU/mL.

C. albicans (SC5314) single colonies were inoculated into 10 mL sterile yeast, peptone, dextrose (YPD) broth and incubated overnight at 30°C at 200 rpm for 16 h. The fungi were suspended with sterile PBS to a final concentration of 3 × 107 CFU/mL.

C. albicans respiratory tract inoculation.

Fungal inoculation was achieved by transglottal inoculation of 3 × 106 CFU C. albicans SC5314 on the first day of experiments. To establish the model, 4 animals each were sacrificed at 4 h, 24 h, and 3, 7, and 14 days after inoculation, and lungs, livers and kidneys were removed. The lungs, livers, and kidneys from one animal were homogenized and used for C. albicans CFU counts, whereas organs from the other three animals were used for histopathologic analyses. We compared lung cytokine levels between C. albicans-inoculated and control rats without A. baumannii infection 48 h after C. albicans or saline inoculations, respectively.

The rat model of A. baumannii infection.

Rats were divided into two groups: a group with prior C. albicans airway inoculation (AWC group; n = 15) and a saline control group (Ab group, n = 15). A suspension of 108 CFU of A. baumannii ATCC 19606 was transglottally inoculated into each animal on the second day of the experiment. At this concentration, A. baumannii pneumonia develops in <50% of immunocompetent rats (19). At 24 h after A. baumannii infection, rats were anesthetized for lung axial CT scans. On the third day, animals were sacrificed. A. baumannii CFU counts, Candida CFU counts, histopathologic analyses, and lung cytokine concentrations were compared between the two groups. Pneumonia was defined as macroscopic and/or microscopic lung inflammation with a bacterial burden of >104 CFU per lung.

Antifungal treatment.

C. albicans-inoculated rats were treated with 1 mg/kg/day of intraperitoneal amphotericin B (AWC+AmpB group; n = 18) or normal saline intraperitoneal injections (AWC group; n = 15) for 3 consecutive days beginning on the day of C. albicans airway inoculation. On the 4th day, rats were infected with A. baumannii as described above, and on the 5th day, they were sacrificed for lung tissue A. baumannii CFU counts, C. albicans CFU counts, and cytokine measurements.

Recombinant IL-17A supplementation.

Rats inoculated with C. albicans prior to A. baumannii infection were divided into two groups: those treated with rIL-17A (catalog no. 8410-IL; R&D Systems, Minneapolis, MN, USA) (AWC+IL-17A group, n = 17) and those receiving no treatment (AWC group; n = 15). Rats were treated with 2 mg/kg rIL-17A per rat by intraperitoneal injection 2 h before and 2 h after A. baumannii infection. At 24 h after A. baumannii infection, the rats were anesthetized for lung axial CT scans or sacrificed to collect BALF and lungs. BALF was collected to quantify immune cell populations, cytokines and chemokines produced, LDH, and bacterial loads. The ROS, phagocytosis, and scavenging capacity of cells in BALF were determined as well. The right lung lobe was prepared by homogenizing the tissue in PBS to determine bacterial and fungal loads, MPO activity, and cytokines and chemokines produced and for Western blot analysis. The left lung lobe was used to prepare slides for histopathological examination. The ROS, phagocytic activity, and bacterial killing ability of cells in BALF were also determined.

BALF and cells.

For BALF collection, 5 mL of PBS was administered to the lungs and then retrieved, and this was repeated 3 times to collect a total of 15 mL of BALF. The collected BALF was centrifuged at 1,200 rpm for 10 min at room temperature. The supernatant was retained to examine LDH. The total number of cells in the BALF was determined using a hemocytometer. Cells (1 × 106) were transferred into a 12-well plate for ROS detection. The remaining cells were suspended in sterile PBS and prepared for flow-cytometric analysis.

Flow-cytometric analysis of BALF cells.

BALF cell suspensions were obtained from the rats. The expression of cell surface markers was determined by flow cytometry using fluorescent dye-conjugated rat antibodies. Rat neutrophils were identified by CD45 and RP-1 expression. Rat alveolar macrophages were identified by CD45 and CD68 expression. To analyze apoptosis, BALF cells were incubated with BV421-labeled annexin V and propidium iodide. Data were acquired with the FACSVerse cell analyzer (BD Biosciences, San Diego, CA, USA) and analyzed with FlowJo software (BD Biosciences, San Diego, CA, USA) (version 7.6).

Bacterial counts in BALF and lungs.

A 100-μL aliquot of BALF or lung homogenate was spread onto LB agar plates. Following overnight incubation at 37°C, bacterial colonies were counted, and the number of bacteria was expressed as CFU per milliliter of BALF or CFU per gram of lung tissue.

Fungal counts in BALF and lungs.

A 100-μL aliquot of BALF or lung homogenate was spread onto YPD agar plates. Following overnight incubation at 30°C, fungal colonies were counted, and the number of fungi was expressed as CFU per milliliter of BALF or CFU per gram of lung tissue.

Histopathologic examination.

Left lung lobes were harvested and fixed in 10% neutral formalin for histopathological examination. The tissues were processed in an alcohol and xylene series and embedded in paraffin using standard procedures. Three-micrometer sections were prepared, stained with hematoxylin-eosin (H&E), and examined under a microscope.

Lung MPO assay.

Lung tissues were frozen at −80°C and homogenized in PBS. Lung tissues MPO activity was determined using MPO Detection Kit (catalog no. A044-1-1; Nanjing Jiancheng Bioengineering Institute, China). Briefly, lung tissues were homogenized in 1 mL of 50 mmol/L potassium containing hexadecyltrimethylammonium hydroxide and centrifuged at 12,000 rpm at 4°C for 20 min. Ten microliters of the supernatant was transferred into PBS (pH 6.0) containing 0.17 mg/mL 3,3′-dimethoxybenzidine and 0.0005% H2O2. MPO activity of the supernatant was determined by measuring the H2O2-dependent oxidation of 3,3′-dimethoxybenzidine and expressed as units per gram of total protein.

Measurement of cytokines and chemokines.

Following the manufacturer’s instructions, concentrations of IFN-γ, TNF-α, IL-1β, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17A, G-CSF, MIP-1α, MIP-2, IL-18, and TNF-α in lung homogenates were determined using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA). In brief, target-specific antibody (2 ng/mL) was used to coat the bottom of the wells of 96-well plates overnight at 4°C. Plates were then washed with Tris-buffered saline with Tween (TBST; 20 mM Tris [pH 7.5], 150 mM NaCl, and 0.05% Tween 20) and blocked for 1 h at room temperature with 1% bovine serum albumin (BSA) in TBST. A standard curve was prepared using recombinant protein standards. Following blocking, samples or standards were added to the wells, incubated for 1 h at room temperature, washed with TBST, and incubated for 2 h at room temperature with biotinylated detection antibody (100 ng/mL). After incubation, plates were washed with PBS (pH 7.4). Plates were then incubated for 20 min at room temperature with streptavidin conjugated to horseradish peroxidase (HRP), washed again, and incubated for 5 min in the dark with 100 μL of 3,3′,5,5′-tetramethyl-benzidine (TMB) (Sigma-Aldrich, St. Louis, MO). Finally, 50 μL of 1 M H2SO4 stop solution was added to quench the reaction, and optical density was measured using a microplate reader (Luminex [Austin, TX] 100 IS system) set at 450 nm.

Migration, phagocytic activity, and bacterial killing ability of neutrophils.

One hundred microliters of cell suspension (2 × 105 cells) was added to the upper chamber of Transwell plates, and 1,000 μL of an A. baumannii suspension at 1/10 the multiplicity of infection (MOI) with or without 0.1 mg/mL of rIL-17A was added to the lower chamber. The Transwell chambers were placed in an incubator with 5% CO2 and incubated at 37°C for 2 h. The number of neutrophils that migrated to the lower chamber was measured with a hemocytometer.

Neutrophil phagocytosis of bacteria was determined using a gentamicin protection assay. In this assessment, neutrophils were seeded into 12-well plates at a density of 1 × 106 cells per well and incubated at 37°C with 5% CO2. The cells were pretreated with or without recombinant IL-17A for 1 h and then infected with A. baumannii at an MOI of 1:10. After incubation for 1 h, gentamicin (5 μg/mL), a non-membrane-permeative antibiotic, was added to the medium for 30 min to eliminate extracellular bacteria. The cells were washed with PBS 1 h after infection (phagocytosis) or 6 h after infection (bacterial killing) and subsequently lysed with 1% Triton X-100 in PBS. Cell lysates were plated on LB agar to determine the number of living bacteria engulfed by neutrophils.

Western blot analysis.

Western blot analysis was performed using lung homogenates. Lungs were lysed with ice-cold radio immunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail [200 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 30 μM aprotinin, 13 mM bestatin, 1.4 mM E64, 1 mM leupeptin] for 30 min. All manipulations were performed on ice. Protein content was determined by using a bicinchoninic acid (BCA) assay. Protein samples (30 μg per lane) were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. Blots were blocked with 5% skim milk powder in TBST. The membranes were incubated with primary antibodies, such as antibodies to MLKL (catalog no. 37705; Cell Signaling Technology, Danvers, MA, USA), pMLKL (catalog no. 37333, Cell Signaling Technology, Danvers, MA, USA), RIPK3 (catalog no. 15828; Cell Signaling Technology, Danvers, MA, USA), Casp1 (catalog no. 83383; Cell Signaling Technology, Danvers, MA, USA), and β-actin (catalog no. 4970; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Then, PVDF membranes were washed with TBST, and anti-rabbit HRP-conjugated secondary antibodies were applied to the membranes for 1 h. The target protein was detected using the LI-COR infrared screen Photoscan.

ACKNOWLEDGMENTS

This work was supported in part by the Division of Intramural Research of the NIAID/NIH (ZIA AI001175 to M.S.L.). This work was supported by the National Natural Science Foundation of China (grant 81570012) to X.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We have no financial disclosures.

Contributor Information

Michail S. Lionakis, Email: lionakism@niaid.nih.gov.

Eleftherios Mylonakis, Email: emylonakis@Lifespan.org.

Xiaojiang Tan, Email: txjzdk@126.com.

Mairi C. Noverr, Tulane School of Medicine

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