Online Laboratory Investigations

Airway Fungal Colonization Compromises the Immune System Allowing Bacterial Pneumonia to Prevail Damien Roux, MD, PhD1,2,3; Stéphane Gaudry, MD1,2,3; Linda Khoy-Ear, MD1,2; Meryem Aloulou, PhD4,5; Mathilde Phillips-Houlbracq, MSc1,2; Julie Bex1,2; David Skurnik, MD, PhD6; Erick Denamur, MD, PhD1,2; Renato C. Monteiro, MD, PhD4,5,7; Didier Dreyfuss, MD1,2,3; Jean-Damien Ricard, MD, PhD1,2,3

Objective: To study the correlation between fungal colonization and bacterial pneumonia and to test the effect of antifungal treatments on the development of bacterial pneumonia in colonized rats. Design: Experimental animal investigation. Setting: University research laboratory. Institut National de la Santé et de la Recherche Médicale, INSERM U722, Paris, France. 2 Univ Paris Diderot, Sorbonne Paris Cité, UMRS-722, Site Xavier Bichat, Paris, France. 3 AP-HP, Hôpital Louis Mourier, Service de Réanimation Médico-chirurgicale, Colombes, France. 4 INSERM UMR-S699, Paris, France. 5 Inflamex Laboratory of Excellence, Paris Diderot University, Sorbonne Paris Cité, Paris, France. 6 Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. 7 Immunology Laboratory, Bichat Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France. This work was performed at Université Paris Diderot. Drs. Gaudry, Khoy-Ear, and Aloulou, and Mr. Phillips-Houlbracq contributed equally. Supported, in part, by the French Society of Intensive Care (SRLF) (Drs. Roux and Phillips-Houlbracq), the Société de Pneumologie de Langue Française (Dr. Gaudry), the Fonds d’Etudes et de Recherche du Corps Médical des Hôpitaux de Paris (Dr. Khoy-Ear), and the Legs Poix (Dr. Ricard). INSERM U722 laboratory received a research grant from Pfizer. Dr. Ricard received grant support from Pfizer; is a board member and lectured for Covidien; and received support for travel from Fisher&Paykel. Dr. Roux received grant support from the French Society of Intensive Care. Dr. Gaudry lectured for Lob Conseils and received support for travel from Issis Medical. Drs. Aloulou and Bex received funding from from INSERM. Dr. Skurnik received grant support from the Hood Foundation and Hearst Foundation. Dr. Monteiro received funding from ANR. Dr. Dreyfuss received grant support from Pfizer. The remaining authors have disclosed that they do not have any potential conflicts of interest. Address requests for reprints to: Jean-Damien Ricard, MD, PhD, Service de Réanimation, Hôpital Louis Mourier–178, rue des Renouillers, 92700 Colombes, France. E-mail: [email protected] 1

Copyright © 2013 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0b013e31828a25d6

Critical Care Medicine

Subjects: Pathogen-free male Wistar rats weighing 250–275 g. Interventions: Rats were colonized by intratracheal instillation of Candida albicans. Fungal clearance from the lungs and immune response were measured. Both colonized and noncolonized animals were secondarily instilled with different bacterial species (Pseudomonas aeruginosa, Escherichia coli, or Staphylococcus aureus). Bacterial phagocytosis by alveolar macrophages was evaluated in the presence of interferon-gamma, the main cytokine produced during fungal colonization. The effect of antifungal treatments on fungal colonization and its immune response were assessed. The prevalence of P. aeruginosa pneumonia was compared in antifungal treated and control colonized rats. Measurements and Main Results: C. albicans was slowly cleared and induced a Th1-Th17 immune response with very high interferon-gamma concentrations. Airway fungal colonization favored the development of bacterial pneumonia. Interferon-gamma was able to inhibit the phagocytosis of unopsonized bacteria by alveolar macrophages. Antifungal treatment decreased airway fungal colonization, lung interferon-gamma levels and, consequently, the prevalence of subsequent bacterial pneumonia. Conclusions: C. albicans airway colonization elicited a Th1-Th17 immune response that favored the development of bacterial pneumonia via the inhibition of bacterial phagocytosis by alveolar macrophages. Antifungal treatment decreased the risk of bacterial pneumonia in colonized rats. (Crit Care Med 2013;41:e191–e199) Key Words: alveolar macrophages; bacterial pneumonia; Candida albicans; interferon-gamma; phagocytosis; Pseudomonas aeruginosa

V

entilator-associated pneumonia (VAP) is a major concern in patients under mechanical ventilation in ICUs (1–3). In spite of intense efforts to control the known risk factors of VAP, these infections remain frequent with high morbidity and possible attributable www.ccmjournal.org

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mortality (4, 5). While bacterial species are responsible for 99% of VAP (1), the role of fungal pathogens is unclear in immunocompetent hosts (6). Candida species are detected in over 50% of ventilated critically ill patients (7, 8), but this microorganism is uncommonly responsible for lung infections in these patients (8–10). Thus, the very existence of true Candida pneumonia in critically ill patients is still questioned (11, 12). However, Azoulay et al (13) observed an association between airway colonization with Candida species and subsequent Pseudomonas aeruginosa VAP, raising the idea that interactions between bacterial and fungal species could be more important than previously considered (14). The latter concept prompted us to investigate a potential interaction between Candida albicans airway colonization and P. aeruginosa pneumonia (15). We observed that while an inoculum of P. aeruginosa caused pneumonia in only 3% of control rats, 34% of rats colonized with C. albicans developed pneumonia. We also noted a 60% decrease in reactive oxygen species production by alveolar macrophages (AMs) in the presence of C. albicans, suggesting an effect of C. albicans on the lung innate immune response. However, a formal causal link between C. albicans colonization and P. aeruginosa pneumonia and/or general bacterial pneumonia required further investigation. Recently, Hamet et al (7, 16) reported Candida colonization as a risk factor for VAP with multidrug resistant bacteria, highlighting the fact that this fungal colonization can no longer be overlooked. Thus, the aims of the present study were 1) to confirm our previous results showing the facilitating effect of C. albicans airway colonization on the development of P. aeruginosa pneumonia and to emphasize this finding by studying two other major bacterial pathogens responsible for VAP (Staphylococcus aureus and Escherichia coli) in the same animal model; 2) to investigate the mechanism of this effect, focusing on bacterial phagocytosis by AMs in the context of a high interferongamma (INF-γ) pulmonary concentration induced by fungal airway colonization; 3) to evaluate the consequences of our findings in a potential clinical situation by assessing the effect of an antifungal treatment on the development of bacterial pneumonia in the context of fungal airway colonization.

MATERIALS AND METHODS Animals Experiments were performed on pathogen-free male Wistar rats weighing 250–275 g in compliance with the recommendations from the French Ministry of Agriculture and approved by the French Veterinary Services. Transglottic instillations of rats were performed as described previously (15, 17). Microorganisms The ATCC 10231 C. albicans, originally isolated from a patient with bronchomycosis, was used in the study. Three bacterial species were used: P. aeruginosa PAO1, S. aureus NCTC 8325, and E. coli P5.21 (B2 phylogenetic group, serogroup O6 [18]). C. albicans (3 × 106 colony-forming unit [CFU]/instillation) e192

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was prepared as described previously (15). Bacteria were grown overnight in Luria Bertani broth, washed with saline, and diluted to the chosen concentration. The inocula of P. aeruginosa (6 × 106 CFU), S. aureus (3 × 107 CFU), and E. coli (2 × 106 CFU) were chosen to induce a bacterial pneumonia in less than 50% of the animals (preliminary data). To induce pneumonia in more than 80% of the animals, an inoculum of 8 × 107 CFU of P. aeruginosa was selected. Fungal Airway Colonization, Immune Response, and Development of Subsequent P. aeruginosa Pneumonia Airway fungal colonization was obtained with a single intratracheal instillation of 3 × 106 CFU of C. albicans. Fungal clearance and the local immune response were assessed over time. Animals were killed 4 hours, 24 hours, 72 hours, 7 days, and 14 days after instillation, and C. albicans count was performed in homogenized lungs to evaluate spontaneous clearance. Levels of interleukin (IL)-2, INF-γ, IL-10, IL-4, and IL-17 in homogenized lungs were measured using a rat-specific ELISA kit (R&D and eBioscience, France) at 24 and 72 hours after the fungal instillation (or a control instillation with saline) to evaluate the Th1, Th2, and Th17 local immune response in the presence of C. albicans. To measure the effect of the fungal airway colonization on the development of bacterial pneumonia, the predetermined bacterial inoculum was instilled 24 or 72 hours after C. albicans, and the lungs were analyzed as described previously (15). A bacterial pneumonia was defined as the co-occurrence of a macroscopic pulmonary inflammation and a bacterial count above 104 CFU per instilled lung (15). Bacterial Phagocytosis by Alveolar Macrophages Phagocytosis was evaluated as described previously (19). Briefly, AMs were recovered from the bronchoalveolar lavage of normal rats and incubated overnight with recombinant rat INF-γ (eBiosciences, Paris, France) at 10 ng/mL (20) or a control solution (phosphate buffered saline + bovine serum albumin 1%). Commercially available Texas red-coupled bacteria (E. coli and S. aureus, Molecular Probes, Leiden, The Netherlands) were individually added at 37°C for 30 minutes (107 bacteria per 5 × 105 cells), with or without previous opsonization. Macrophage membranes were stained with CD11b-FITC on ice for 20 minutes before confocal microscopy. Images were taken with a LSM 510 confocal microscope (Carl Zeiss, Le Pecq, France). The intensity of the red fluorescence inside each AM was quantified. Antifungal Treatment Two antifungal treatments were chosen: fluconazole and amphotericin B. The minimum inhibitory concentrations of the C. albicans strain were tested by e-test (AB Biodisk, Solna, Sweden). For in vivo experiments, the daily doses were 10 mg/ kg for fluconazole (21) and 1 mg/kg for amphotericin B (22, 23) through intraperitoneal injection. Treatment or control (saline solution) doses were started immediately after the fungal instillation and for the following 2 days. The effect of antifungal treatment or control was assessed 72 hours after September 2013 • Volume 41 • Number 9

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fungal instillation through quantitative fungal cultures and measuring INF-γ levels in the lungs. Before testing an antifungal treatment on the development of P. aeruginosa pneumonia in colonized animals, we evaluated its direct effect on the development of the same pneumonia in the absence of C. albicans. Noncolonized rats were similarly treated; after 3 days, the P. aeruginosa inoculum (8 × 107 CFU) induced pneumonia in most of the rats. To test the effect of the antifungal treatment on P. aeruginosa pneumonia during fungal airway colonization, the bacteria (6 × 106 CFU) were instilled 72 hours after C. albicans (24 hr after the third and last dose of antifungal treatment) and the lungs were evaluated 24 hours after the bacterial challenge. Statistics The results are presented as median (interquartile range [IQR] 25; IQR, 75). The statistical analyses were performed with GraphPad Prism 4.00. Categorical data were compared by the chi-square test or Fisher exact test when an expected value was less than five. Continuous variables were compared using the Mann-Whitney U test for two groups, or Kruskal-Wallis test for more than two groups with the Dunn’s multiple comparison posttest if statistical significance was found. A p value of less than 0.05 was considered significant.

RESULTS Fungal Colonization Facilitates the Development of Subsequent P. aeruginosa Pneumonia A new simplified model of fungal airway colonization was tested and found to be responsible for an increased prevalence of P. aeruginosa pneumonia, confirming our previous observation (15). In this model, instead of three successive fungal instillations of a clinical strain, fungal airway colonization was obtained through a single instillation of a laboratory strain of C. albicans. C. albicans Colonization.  After intratracheal instillation, C. albicans was slowly cleared from the lungs (Fig. 1A) while the body weights of the colonized rats increased, similar to the uninstilled animals (Fig. 1B). As previously reported (15), only rare spots of peribronchial inflammation were caused by the Candida colonization (data not shown).

P. aeruginosa Pneumonia. Twenty-four hours after fungal colonization, instillation of the well-described laboratory strain P. aeruginosa PAO1 (24) induced significantly more bacterial pneumonia as compared to control rats (14 of 32 vs 5 of 32, respectively; chi-square: p < 0.05, Table 1). An example of the macroscopic aspect of pneumonia is presented in Figure 2. Bacterial counts and lung weights were significantly higher in the colonized group as compared to the control group (MannWhitney U test: p < 0.05 for each; Fig. 3A). Because high levels of C. albicans were still present in the lungs, 72 hours after the fungal instillation (Fig. 1), we tested a delayed P. aeruginosa instillation at 72 hours. Again, an increased prevalence of P. aeruginosa pneumonia (13 of 31 vs 6 of 32 in the colonized group and the control group, respectively; chi-square: p < 0.05; Table 1) was observed. In addition, the bacterial counts tend to be higher in the colonized group, and lung weights significantly differed between the two groups (Mann-Whitney U test: p = 0.06 and p < 0.05, respectively; Fig. 3B). Effect of Fungal Airway Colonization on Other Bacterial Pneumonia (S. aureus and E. coli) After confirmation of the facilitating effect of fungal airway colonization on the development of P. aeruginosa pneumonia with a simplified model of colonization, we investigated whether similar effects could be obtained with two other major bacterial pathogens involved in lung infection: S. aureus and E. coli (1, 2). Both microorganisms were individually instilled 24 hours after the fungal inoculum; bacterial pneumonias were more frequent in the colonized groups than in controls (14 of 15 vs 8 of 18 for S. aureus, chi-square: p < 0.01, and 6 of 20 vs 0 of 20 for E. coli, Fisher exact test: p < 0.05; Table 1). As previously shown, bacterial counts and lung weights were higher in the fungal colonized groups (Mann-Whitney U test: p < 0.001 and p < 0.01 for S. aureus and E. coli, respectively; Fig. 3, C and D).

Fungal Airway Colonization and Immune Response in Colonized Lungs To understand the mechanism linking fungal colonization to the development of subsequent bacterial pneumonia, we decided to study the immune response associated with fungal airway colonization. As shown previously, after a single intratracheal instillation of C. albicans, the fungus was slowly cleared from the lungs over time. This airway colonization was found to be associated with significant increases in the levels of Th1specific INF-γ and Th17-specific IL-17 whereas the Th2-specific IL-10 and IL-4 levels remained unchanged (Kruskal-Wallis test: p < 0.001, p < 0.05, p = 0.49, and p = 0.07, respectively; Fig. Figure 1. Airway colonization with Candida albicans. Rats were intratracheally instilled with C. albicans 4). Conversely, IL-2 decreased (3 × 106 colony-forming unit [CFU]). At different time points, animals were killed and lungs homogenized. significantly (Kruskal-Wallis test: A, Fungal count was determined at 4 hr, 24 hr, 72 hr, 7 days, and 14 days after instillation. B, Body weight p < 0.01; Fig. 4). At 24 hours, this variation was compared at each time point with normal rats. Median and interquartile range are represented. Critical Care Medicine

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Table 1.  Prevalence

of Bacterial Pneumonia in Colonized and Control Rats No. With Pneumonia/No. of Rats Time After Colonization (hr)

C. albicans Airway Colonization

Control

p (Chi-Square or Fisher Exact Testa)

Pseudomonas aeruginosa

24

14/32

5/32

< 0.05

P. aeruginosa

72

13/31

6/32

< 0.05

Escherichia coli

24

6/20

0/20

< 0.01a

Staphylococcus aureus

24

14/15

8/18

< 0.01

Bacterial Strain

cytokine profile of increased INF-γ and IL-17 and decreased IL-2, when compared to the control group with Dunn’s post hoc test (p < 0.001, p < 0.01, and p < 0.05, respectively), was consistent with a Th1-Th17 immune response. At 72 hours, only a tendency to an increased INF-γ level was observed (Fig. 4).

to recognize the bacteria and bypassed the need for scavenger receptors, allowed efficient phagocytosis in the presence of INF-γ with a 22% increase in the mean intracellular fluorescence when opsonized E. coli were used (Mann-Whitney U test, p < 0.05; Fig. 5, A and B).

Phagocytosis by Alveolar Macrophages in the Presence of INF-γ Because high levels of INF-γ lead to a decrease in the expression of scavenger receptors on AMs (20, 25), the major increase in INF-γ levels observed after fungal colonization led us to the hypothesis that an inhibition of the bacterial phagocytosis by AMs could explain the in vivo results. In the absence of stable fluorescent P. aeruginosa strain, commercially available red-labeled E. coli and S. aureus were tested in an in vitro model of bacterial phagocytosis (19) by AMs in the presence or absence of INF-γ. Following an overnight culture with INF-γ, the phagocytosis of unopsonized bacteria (red-fluorescent E. coli and S. aureus) by AMs (green-fluorescent membrane) was markedly decreased (Fig. 5A). In fact, the mean intracellular fluorescence (red fluorescence measured inside the green membrane) was decreased by more than 90% in the presence of INF-γ as compared to control AMs (Mann-Whitney U test, p < 0.0001 for E. coli and S. aureus; Fig. 5B). As expected, opsonization of the bacteria, which allowed the Fcγ receptors

Effect of Antifungal Treatment on C. albicans Airway Colonization and Subsequent P. aeruginosa Pneumonia After studying the mechanism of action of fungal colonization on bacterial pneumonia, we investigated the effect of antifungal therapy on 1) the fungal count in the lung, 2) the local immune response raised during the course of the colonization, and 3) its potential to prevent bacterial pneumonia among colonized animals. The strain of C. albicans was susceptible to fluconazole and amphotericin B with minimum inhibitory concentrations equal to 0.50 and 0.047 µg/mL, respectively. In vivo, both treatments decreased fungal counts in lungs in comparison to the control group (Kruskal-Wallis test, p < 0.001; Fig. 6A) with a greater effect observed with amphotericin B than fluconazole (Dunn’s post hoc test, p < 0.01). Interestingly, the pulmonary levels of INF-γ were dramatically decreased by antifungal treatments as compared to the control group (Kruskal-Wallis test, p < 0.01; Fig. 6B). INF-γ levels in the treated groups did not differ from those in noncolonized rats (Dunn’s post hoc test, p > 0.05 between each treated groups and noncolonized group).

Figure 2. Macroscopic aspects of Pseudomonas aeruginosa instilled lungs. A, Normal macroscopic aspect of lungs from a noncolonized rat that received 6 × 106 colony-forming unit (CFU) of P. aeruginosa PAO1 and did not develop pneumonia. B, Macroscopic aspect of lungs colonized with Candida albicans that develop pneumonia after an instillation of the same bacterial inoculum as in A. Note the marked hepatization of one pulmonary lobe.

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Figure 3. Effect of airway colonization by Candida albicans on the development of bacterial pneumonia. At day 0, anesthetized rats received either C. albicans (3 × 106 colony-forming unit [CFU], colonized group, closed squares and circles) or saline (control group, open squares and circles) in the trachea. Then a bacterial instillation was performed in a main bronchus: Pseudomonas aeruginosa (6 × 106 CFU) was instilled either 24 or 72 hr after the fungus (panel A or B, respectively); Escherichia coli (3 × 107 CFU, panel C) or S. aureus (2 × 106 CFU, panel D) were instilled 24 hr after the fungus. Rats were killed 24 hr after the bacterial instillation and lungs were aseptically removed. Bacterial counts (square) and lung weights (circle) were significantly higher in colonized groups (black) as compared to respective control groups (open). Bars represent the median. Mann-Whitney test: *p < 0.05; **p < 0.01; ***p < 0.001. Results are from at least two independent experiments.

In contrast, IL-17 concentrations were not significantly affected by antifungal treatments (Kruskal-Wallis test, p = 0.92; Fig. 6C). Finally, we tested the effect of antifungal treatment in our model of P. aeruginosa pneumonia after fungal colonization. First, to avoid any direct effects of the antifungal treatment on P. aeruginosa pneumonia, we tested this treatment in a model of P. aeruginosa pneumonia in noncolonized animals using a higher bacterial inoculum. In the absence of fungal colonization, the antifungal treatment had no effect on the development of P. aeruginosa pneumonia with 100% (10 of 10) pneumonia in the treated group and 90% (9 of 10) in the control group (Fisher exact test, p = 1). The bacterial counts and lung weights did not differ between the treated and control groups (57,600 [37,750; 104,000] vs 46,000 [20,200; 80,650] CFU/instilled lung, MannWhitney U test: p = 0.35, and 1,917 [1,827; 1,993] vs 1,838 [1,673; 2,048] mg, Mann-Whitney U test: p = 0.53, respectively). Critical Care Medicine

Second, in fungal-colonized animals, the antifungal treatment decreased the prevalence of P. aeruginosa pneumonia as compared to the untreated group (7 of 47 vs 15 of 46, respectively, chi-square: p < 0.05). Again, bacterial counts and lung weights were significantly lower in the treated group (Mann-Whitney U test: p < 0.05 and p < 0.001, respectively; Fig. 6D).

DISCUSSION This report confirms the increased prevalence of murine P. aeruginosa pneumonia observed in the context of C. albicans airway colonization (15). It extends this observation to two other important bacterial pathogens: S. aureus and E. coli; these three groups of bacterial pathogens account for up to 70–80% of the pathogens responsible for VAP (26, 27). As one possible contributing factor, we found that the high INF-γ www.ccmjournal.org

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Figure 4. Cytokine levels in lungs before and after colonization with Candida albicans. Lungs of control rats or rats colonized with C. albicans (3 × 106 colony-forming unit [CFU] intratracheally) were homogenized. Interferon (INF)-γ, interleukin (IL)-2, IL-17, IL-10, and IL-4 concentrations were measured 24 and 72 hr after fungal colonization or a saline instillation (control). Median and interquartile range are represented. Kruskal-Wallis test: *p < 0.05; **p < 0.01; ***p < 0.001.

concentration observed in the lungs during fungal airway colonization inhibited bacterial phagocytosis by AMs. In addition, we showed that an antifungal treatment was able to normalize INF-γ levels and remove the facilitating effect of fungal colonization on bacterial pneumonia in vivo. We hypothesize that the presence of C. albicans in the airways could induce an immune response that may in turn decrease the normal antibacterial function of the immune cells, thus allowing bacterial pathogens to initiate an actual infection instead of being cleared by the immune system. In this work, we first developed a simplified model of airway colonization with a single instillation of C. albicans. This model fulfilled the predefined criteria of colonization, with no signs of patent infection (only mild airway inflammation was observed histologically, data not shown) and slow fungal clearance from the lungs (Fig. 1A), while the body weights of the colonized rats increased similarly to normal rats (Fig. 1B). This fungal airway colonization was responsible for a Th17 immune response with increased levels of IL-17 in the lungs and a decrease in IL-2, a cytokine known to repress the Th17 response (28). We also observed a very significant increase in the production of INF-γ, which is a major cytokine of the Th1 lineage. Interestingly, increased levels of INF-γ have recently been associated with a Th17 immune response (29–32). INF-γ could be secreted by the AMs themselves (33), by natural killer T cells (34), and also by INF-γ/IL-17 double producer T cells (29, 31, 32). The absence of increases in IL-4 and IL-10 concentrations in the lungs indicated that the Th2 immune response was totally absent in our model. Thus, our model of fungal colonization induced a Th1-Th17 immune response with very high levels of INF-γ, as observed in other murine model of candidiasis (35) and in ex vivo human experiments (32). Interestingly, this Th1-Th17 combined immune response, which arose after immunization with the candidal Als3p adhesin (rAls3p-N), is required to protect against C. albicans (30). AMs represent the primary immune cell line against inhaled pathogens that reach the alveoli. Because bacteria are e196

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not initially opsonized by specific antibodies, internalization of the pathogen by professional phagocytes relies on scavenger receptors (36, 37). It has been shown that the expression of the major scavenger receptor of AMs (macrophage receptor with collagenous structure [38, 39]) decreases in the presence of INF-γ in both mice (20) and humans (25). Keeping in this line of reasoning, our finding of an increase in INF-γ levels, induced by fungal colonization, is interesting. Indeed, our in vitro model of phagocytosis showed that INF-γ strongly inhibited bacterial phagocytosis by AMs when the bacteria were not opsonized by specific antibodies. Thus, our results are in accordance with decreased AM phagocytosis due to a decrease in the expression of the AM scavenger receptors after activation by INF-γ as previously shown (20). Conversely, we observed an increase in bacterial phagocytosis by INF-γ-stimulated AMs after opsonization of the bacteria. This was expected because the expression of Fcγ receptors (especially Fcγ-R1), which bind opsonized bacteria, increases after INF-γ stimulation (40). This finding complements the previous observation that C. albicans could modulate the innate immune response mediated by AMs through a decreased production of reactive oxygen species (15). The antifungal treatment of the colonized animals significantly decreased the risk for P. aeruginosa pneumonia in treated rats as compared to untreated colonized animals. While examining the mechanism for this result, we noticed a significant decrease in the INF-γ concentration in the lungs of treated rats. This observation confirmed the correlation between the increased production of INF-γ and an increased risk of bacterial pneumonia. The absence of IL-17 decrease after antifungal treatment underlines the complex interaction between fungal antigens and immune receptors, and thus may highlight the multiple immune pathways responsible for cytokine secretion after fungal recognition (41). Our main hypothesis for the increased prevalence of murine bacterial pneumonia in the context of C. albicans airway colonization is based on a three-step mechanism. First, a Th1-Th17 immune response with an increase in the production of IL17 and INF-γ is induced by C. albicans colonization, without activation of the Th2 lineage. Second, as a consequence, the host antibacterial defense is impaired by the increased levels of INF-γ, with concurrent inhibition of the phagocytosis of nonopsonized bacteria by AMs, possibly through a decreased expression of scavenger receptors. Finally, these bacteria that have evaded the host immune defenses can more readily provoke pneumonia. Once again, this remains a hypothesis. It is however in agreement with the murine model developed by Sun and Metzger (20). They found an increased prevalence of Streptococcus pneumoniae pneumonia following a marked Th1 immune response with high levels of INF-γ observed during a Myxovirus influenzae infection in mice. Furthermore, these authors showed that INF-γ neutralization had a protective effect on the development of S. pneumoniae pneumonia, similar to the protective effect we observed during the antifungal treatment that normalized INF-γ levels in the lungs (Fig. 6). In contrast to our results, a beneficial effect of short-term airway colonization by C. albicans on P. aeruginosa lung infection was recently reported (42). Interestingly, this in vivo model was developed with BALB/c mice. Contrary to our rat model, these September 2013 • Volume 41 • Number 9

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of C. albicans airway colonization apart from our initial one (15). Hence, it was difficult for us to set the exact timing between Candida instillation and secondary bacterial challenge. We adjusted our initial model by increasing the duration of Candida colonization so as to approximate the clinical setting. Second, to comply with ethical regulations on limitation of animal experimentation, we did not repeat experiments performed in our previous article clearly indicating that the observed increase in susceptibility to bacterial infection was not a nonspecific inflammatory response (15). Indeed, administration of killed C. albicans elicited no inflammatory response and no increase in bacterial count. A reasonable proportion of the colonized population showed CFU results similar to the noncolonized group (Fig. 3). As we were using outbred rats (Wistar), a possible explanation was a differential host response within animals. However, the cytokine concentrations were homogeneously distributed within the colonized group, suggesting certain homogeneity Figure 5. Effect of interferon-gamma (INF-γ) on bacterial phagocytosis by alveolar macrophages. Alveolar in the host response. macrophages recovered from bronchoalveolar lavage of normal rats were incubated with recombinant rat INF-γ or its control. After incubation with Texas Red-coupled bacteria (red-labeled unopsonized Escherichia coli and Furthermore, as the bacteria Staphylococcus aureus, or opsonized E. coli), cell surface was stained in green with a biotin primary antibody were instilled in a main anti-rat CD11b. Images were taken with a confocal microscope. A, In the absence of INF-γ, unopsonized bronchus, pneumonia was or opsonized bacteria were internalized (red fluorescence inside green-stained cell surface). After INF-γ incubation, alveolar macrophages were unable to internalize unopsonized bacteria. This was not the case after predominantly unilateral. Thus, bacterial opsonization. B, Mean red fluorescence intensity (MFI) inside green-stained alveolar macrophages was the volume of the infected dramatically decreased after overnight culture with INF-γ when unopsonized E. coli or unopsonized S. aureus lung was frequently modest as were used. Inversely, INF-γ slightly increased MFI when using opsonized E. coli. Mann-Whitney test: *p < 0.05; ***p < 0.001. Median and interquartile range are represented. compared to the whole lung volume that was analyzed, mice characteristically develop a Th2 immune response with resulting in a moderate increase in the CFU count of animals increased IL-4 and IL-10 cytokines after intranasal instillation developing pneumonia. Finally, we observed a greater effect or mucosal challenge with C. albicans (43, 44). The pattern of of fungal colonization on the development of pneumonia for cytokine production during fungal colonization thus appears both E. coli and S. aureus as compared to P. aeruginosa (Fig. 3), to be animal model dependent. In our study, we observed a this could be due to the low virulence of the PAO1 strain in our specific Th1-Th17 response, the same immune response that model. Indeed, the well-described laboratory strain PA01 does protects against candidiasis in humans (32, 45–47). not produce the effector protein ExoU, a major virulence factor in P. aeruginosa lung infection (24). Strength and Limits Our results provide a plausible explanation for the assoThere is very limited data on the kinetics of Candida airway ciation between airway colonization with Candida species and colonization in ICU. In addition, there is no published model subsequent P. aeruginosa VAP in ICU patients (13): a reaction Critical Care Medicine

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Figure 6. Effect of antifungal treatment on airway fungal colonization and on Pseudomonas aeruginosa pneumonia. All rats were colonized with an intratracheal instillation of Candida albicans (3 × 106 colony-forming unit [CFU]). They received amphotericin B, fluconazole, or placebo daily by intraperitoneal injection just after fungal instillation and during two more days. A, Twenty-four hours after the third IP injection, fungal counts were significantly decreased by antifungal treatments with a greater effect of amphotericin B (Kruskal-Wallis test, p < 0.0001, Dunn’s multiple comparison test). B, Lung levels of interferon-gamma (INF-γ) decreased significantly and were not different from levels observed in noncolonized rats (Kruskal-Wallis test, p < 0.01, Dunn’s multiple comparison test). C, Interleukin (IL)-17 lung levels did not exhibit any significant differences with or without antifungal treatments (Kruskal-Wallis test, p = 0.82). D, Colonized animals that received either three daily doses of amphotericin B or control by intraperitoneal injection were instilled with P. aeruginosa (6 × 106 CFU) into a main bronchus 24 hr after the third injection. Rats were killed 24 hr after the bacterial instillation and lungs evaluated. Bacterial counts (squares) and lung weights (circles) were significantly lower in the treated group (gray) as compared to the control group (black, Mann-Whitney test). Bars represent the median on A and D; median and interquartile range are represented on B and C. *p < 0.05; **p < 0.01; ***p < 0.001.

of the host immune system to the presence of the nonpathogenic Candida leading to a reduced ability to clear pathogenic bacteria. The finding that antifungal treatment normalized INF-γ levels in colonized airways and allowed the host immune system to efficiently protect against subsequent bacterial challenge is in concurrence with the interesting results of Nseir et al (48), who found a link between antifungal treatment and a decreased prevalence of P. aeruginosa pneumonia among ventilated patients with airway colonization by Candida spp. Overall, our results may have some clinical consequences, given the high proportion of ventilated ICU patients with C. albicans airway colonization (7, 8) and the growing concern regarding the impact of this colonization on bacterial pneumonia (7, 13, 48).

CONCLUSIONS Our results suggest that C. albicans airway colonization could facilitate the development of bacterial pneumonia. In vivo, this effect was blunted by antifungal treatment. A possible e198

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mechanism for these findings is the inhibition of AM phagocytosis in relation with a Th1-Th17 immune response triggered by the fungal colonization.

ACKNOWLEDGMENTS We thank Gerald B. Pier for reviewing the manuscript.

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