Background
Group IVA cytosolic phospholipase A
2 (cPLA
2α) releases arachidonic acid to initiate eicosanoid production [
1]. Eicosanoids are secreted and act locally through G-protein coupled receptors, which are expressed in a cell-type specific manner and initiate distinct signaling pathways to promote diverse biological responses [
2‐
4]. Arachidonic acid is metabolized by 5-lipoxygenase (5-LO) to leukotrienes, and by constitutive cyclooxygenase (COX)-1 and inducible COX-2 to prostaglandins and thromboxane [
5,
6]. Leukotrienes are pro-inflammatory mediators produced by macrophages, dendritic cells, mast cells, basophils and eosinophils that regulate cell trafficking, cytokine production, vascular permeability and phagocyte function [
7]. The cysteinyl leukotrienes including leukotriene C
4, leukotriene D
4 and leukotriene E
4 are bronchoconstrictors involved in asthma and allergic responses [
2]. cPLA
2α and COXs are widely expressed reflecting the ability of most cells and tissues to produce prostanoids, which have diverse functions [
1,
6]. Prostaglandins regulate normal physiological processes such as female reproduction, hemostasis, kidney function and the maintenance of the gastrointestinal tract [
1]. Although prostaglandins promote acute and chronic inflammation in response to tissue injury they also play a role in the resolution of inflammation and can be anti-inflammatory and immunosuppressive [
8‐
10]. Therefore cPLA
2α mediates the release of arachidonic acid for the production of numerous bioactive lipid mediators that have diverse effects [
1]. This makes its role in regulating responses to infection difficult to predict and would be influenced by the specific tissue involved and nature of the microorganism.
Eicosanoids are produced rapidly in response to engagement of pattern recognition receptors by microbial pathogens and modulate immune cell function by affecting phagocytosis, microbial killing, chemotaxis and the transcriptional program [
7,
10,
11]. We have used resident tissue macrophages from the peritoneal cavity and the lung to study the mechanisms of cPLA
2α activation by the fungal pathogen
Candida albicans [
12‐
16]. Resident tissue macrophages are sentinel cells that are first responders to microbial invasion for initiating host defense to infection [
17]. In resident peritoneal macrophages, activation of cPLA
2α by
C. albicans involves engagement of fungal cell wall polysaccharides β-glucan and mannans to C-type lectin receptors dectin-1 and dectin-2, respectively [
13,
14]. These receptors act with MyD88-dependent pathways to activate cPLA
2α, which involves calcium-induced translocation to membrane and phosphorylation by mitogen-activated protein kinases. In peritoneal macrophages,
C. albicans stimulates an autocrine loop involving cPLA
2α activation, production of prostaglandins and increases in cAMP that affects expression of genes involved in host defense and to dampen inflammation [
15,
16]. In contrast, alveolar macrophages exhibit distinct properties since
C. albicans poorly stimulates cPLA
2α-mediated arachidonic acid release, however, priming with granulocyte macrophage colony-stimulating factor (GM-CSF) enhances arachidonic acid release by increasing expression of dectin-1 [
12].
The lung has several mechanisms to clear environmental triggers that are continuously inhaled to prevent excess inflammation and tissue injury that may compromise gas exchange function [
18]. Candida is the predominant fungal genus in the oral cavity, and dispersal of microoganisms from this site to the lung is a mechanism for shaping the lung microbiome [
19,
20]. Despite potential exposure from the oral cavity, levels of
C. albicans in the healthy lung are low indicating mechanisms for efficient clearance to prevent colonization [
21,
22].
C. albicans is a commensal of mucosal surfaces that does not cause infection unless the immune system is compromised [
23,
24]. Candida lung infection occurs in the critically ill, in patients with cancer and cystic fibrosis, during organ transplantation and in immune compromised individuals [
21,
25,
26]. By comparing cPLA
2α
+/+ and cPLA
2α
−/− mice, we found that cPLA
2α contributes to innate immune defenses in the lung for protection against
C. albicans infection.
Methods
Materials
Hank’s Balanced Salts Solution was from Invitrogen (Carlsbad, CA). ELISA kits were from eBioscience (San Diego, CA) (IL1α, IL1β, TNFα, IL6), from Immunology Consultants Laboratory Inc. (Portland, OR) (albumin), from R&D Systems (Minneapolis, MN) (CCL20) and from PeproTech (Rocky Hill, NJ) (CXCL1, CSF2, CSF3). Antibodies for flow cytometry analysis were from eBioscience (San Diego, CA) (anti-mouse CD45 eF450, CD11c PE, CD24 FITC, CD11b APC, MHC-II I-A/E PerCP-eF710, CD103 FITC) and from BD Biosciences (San Jose, CA) (anti-mouse Siglec F-PE and Ly6G (clone 1A8)-PE). QuickIII staining kit for cytospins was obtained from Astral Diagnostics, NJ. Butylated hydroxytoluene and indomethacin were from Fisher Scientific. Percoll, collagenase XI, Trypsin inhibitor, DNase I, RBC lysis solution were from Sigma-Aldrich (St. Louis, MO). Nylon cell strainers (70 μm) were from BD Biosciences (San Jose, CA). Qiasol lysis reagent, RNeasy Mini Kits and Mouse Cytokines & Chemokines RT2 Profiler PCR Array were from Qiagen (Valencia, CA). Paraformaldehyde was from Electron Microscopy Sciences (Hatfield, PA). XTT Cell Viability Kit was from Cell signaling.
Mice
cPLA
2α
−/− mice were generated as previously described [
27], and backcrossed onto a Balb/c background for 10 generations. Balb/c control mice (cPLA
2α
+/+) were obtained from Charles River (San Diego, CA). Mice were housed under specific pathogen free conditions and used between 8–14 weeks of age. Male mice were used for all experiments with exception as noted in the figure legend. The work with mice was approved by the Institutional Animal Care and Use Committee (IACUC) at National Jewish Health and conducted in accordance with their guidelines.
C. albicans challenge
C. albicans (ATCC SC5314) was grown in YPD medium overnight (30 °C), washed, suspended in endotoxin-free PBS then counted. Counts correlated directly with colony forming units (CFU).
C. albicans was administered by intratracheal instillation to cPLA
2α
+/+ and cPLA
2α
−/− Balb/c mice under isoflurane anesthesia. The trachea was intubated with a gavage needle to instill (50 μl)
C. albicans (10
6–10
7 CFU) or endotoxin-free PBS. Mice were euthanized by CO
2 asphyxiation or cervical dislocation with similar results.
C. albicans expressing green fluorescent protein (GFP) was kindly provided by Dr. Robert Wheeler, The University of Maine. It was generated from the wild type SC5314 strain and exhibits similar virulence as the wild type strain in mice [
28].
Bronchoalveolar lavage
Lungs were lavaged 5 times as described [
12]. For analysis of eicosanoids in bronchoalveolar lavage fluid (BALF), the lavage solution also contained 5 μM indomethacin and 50 μM butylated hydroxytoluene. Cells in lavage were differentiated on cytospins. Albumin, cytokines and chemokines were measured in BALF by ELISA.
Fungal burden
Blood was drained by cutting the inferior vena cava, and then lungs and kidneys were removed asceptically, weighed and homogenized (Omni Tissue Homogenizer, Omni International) in sterile phenol red-free HBSS. Homogenates were serially diluted, plated on Sabouraud dextrose agar plates containing penicillin and streptomycin, and then C. albicans CFU determined after 48 h incubation at 37 °C.
Histology
Lungs were fixed by inflation (1 ml), immersed in formalin (10 %) then dehydrated and embedded in paraffin. Sections (5 μm) were stained with H & E.
Real-time PCR
Lungs from cPLA
2α
+/+ and cPLA
2α
−/− mice were homogenized with an Omni Tissue Homogenizer in Qiasol lysis reagent and RNA isolated using on-column DNase treatment. RNA concentration and purity were determined by UV spectrophotometry, and RNA integrity verified using an Agilent Bioanalyzer 2100. cDNA was synthesized from RNA (200 ng) using RT
2 First Strand Kit (Qiagen). Real-time PCR was performed using RT
2 qPCR Mastermix and a Mouse Cytokines & Chemokines RT
2 Profiler PCR Array according to the manufacturer's protocol using the StepOnePlus Real-Time PCR System (Applied Biosystems). RT
2 PCR arrays in a 96-well format were used containing pre-validated primers tested for efficiency (Qiagen). The RT
2 Profiler PCR Array System included a reverse transcription control preloaded into the primer buffer of the RT
2 First Strand cDNA synthesis kit that measured the relative efficiency of the reverse transcription for all the samples. A genomic DNA control and a positive PCR control were also included in the system. The RT
2 Profiler PCR Array data were normalized to the housekeeping gene
Gusb and the relative gene expression level (2^(−ΔC
t) was calculated using the formula ΔC
t = C
t (gene of interest)- C
t (housekeeping gene). The data were analyzed on the PCR array data analysis SA Biosciences web portal (
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).
Real-time PCR was also performed with cDNA synthesized with random hexamer primers (Fermentas Maxima First Strand cDNA Synthesis Kit, Thermo Scientific) using TaqMan fast universal PCR master mix. TaqMan assay probes used were:
Clec7a (dectin-1) (Mm01183349_m1),
Clec4n (dectin-2) (Mm00490934_m1) and
Gusb (Mm01197698_g1). The housekeeping gene
Gusb was used for normalization. Threshold cycle values (
C
T
) were determined and used for ∆∆C
T analysis of gene expression [
29].
Lung digestion and flow cytometry analysis
After performing bronchoalveolar lavage, blood was drained from the lungs by cutting the inferior vena cava. Lungs were removed, cut into small pieces followed by digestion with 5 ml collagenase solution (0.5 mg/ml collagenase XI, 0.2 mg/ml trypsin inhibitor, 5 % FBS in minimum essential medium) for 1 h at 37 °C with occasional mixing. The digested lungs were sheared with an 18-gauge needle, treated with 50 μl of DNase I solution (5 mg/ml) and then incubated for 10 min at 37 °C. Lung digests were filtered through 70-μm nylon cell strainers and the single cell suspension treated with RBC lysis solution. Cells were counted using a Countess cell counter (Invitrogen, Carlsbad, CA) excluding dead cells with trypan blue. Cells were resuspended in flow cytometry (FC) buffer (2 % FBS, 0.1 % BSA, 0.05 % sodium azide in PBS) at 2 × 10
6 cells/ml. All the steps were done at 4 °C. Cells were dispensed (0.5 x 10
6 cells in 250 μl) in V-shaped 96 well plates. After centrifugation at 1500 rpm for 5 min, the supernatant was removed and 50 μl of FcBlock (anti-CD16/CD32, clone 2.4G2, 40 μg/ml in FC buffer, eBiosciences) was added followed by incubation on ice for 15 min. Cells were then treated with 50 μl of antibody cocktails, incubated on ice for 30 min followed by addition of 150 μl FC buffer then washed in FC buffer. Cells were fixed with 4 % paraformaldehyde in PBS (100 μl/well), pH 7.4, then transferred to FC tubes in 300 μl FC buffer and stored in the dark at 4 °C until analysis. Data were acquired on a Dako Cyan ADP flow cytometer. Compensation and data analyses were performed using FlowJo software (TreeStar, Ashland, OR). After the exclusion of doublets and debris, immune cells were identified by CD45 positive staining. A sequential gating strategy was used to identify cell populations: alveolar macrophages (CD45
+ CD24
− CD11b
− SiglecF
+); tissue macrophages (CD45
+ CD24
− CD11b
+); neutrophils (CD45
+ CD11b
+ Ly6G
+) and CD11b
+ dendritic cells (CD11b
+ DCs) (CD45
+ MHCII
+ CD11c
+ CD11b
+) (Additional file
1) [
30].
Eicosanoid measurements
BALF stored at −80 °C was thawed and mixed with an equal volume of cold methanol. Just before analysis, the samples were diluted in water to a final methanol concentration of less than 15 % and then extracted using a solid phase extraction cartridge (Strata Polymeric Reverse Phase 60 mg/ml; Phenomenex, Torrance, CA). The eluate (1 ml of methanol) was dried and reconstituted in 75 μl of high-performance liquid chromatography (HPLC) solvent A (8.3 mM acetic acid buffered to pH 5.7 with NH
4OH) and 25 μl of solvent B (acetonitrile/methanol, 65/35, v/v). An aliquot of each sample (30 μl) was injected into an HPLC and metabolites separated on a C18 column (Kinetex EVO C18 100A 50 x 3.0 mm, 5 μm; Phenomenex, Torrance, CA) eluted at a flow rate of 0.25 ml/min with a linear gradient from 25 % to 75 % solvent B in 13 min then increased to 98 % in 2 min and held for 11 min. The HPLC system was directly interfaced into the electrospray ionization source of a triple quadrapole mass spectrometer (Sciex API 5500; PE-Sciex, Thornhill, ON, Canada). Mass spectrometric analyses were performed in the negative ion mode using multiple reaction monitoring of the specific transitions: [d4]PGE
2
m/z 355 → 275, [d4]PGD
2
m/z 355➔237, [d4]TXB
2
m/z 373 → 173, [d4]6-keto-PGF
1α
m/z 373➔167, [d5]LTC
4
m/z 629 → 271, [d5]LTD
4
m/z 500➔177, [d5]LTE
4
m/z 443➔338, PGE
2
m/z 351 → 271, PGD
2
m/z 351➔233, TXB
2
m/z 369 → 169, 6-Keto-PGF
1α
m/z 369➔ 163, LTC
4
m/z 624 → 272, LTD
4
m/z 495➔177, LTE
4
m/z 438➔333. Quantitation was performed using a standard isotope dilution curve as described [
31].
C. albicans recognition and killing assays
Alveolar macrophages were isolated from untreated cPLA
2α
+/+ and cPLA
2α
−/− mice by lavage and cultured as previously described [
12]. Live opsonized and unopsonized GFP-
C. albicans (moi 2) was used for all assays. GFP-
C. albicans was opsonized by incubating in DMEM containing 10 % mouse serum for 30 min at 37 °C before incubation with the macrophages. For evaluating binding and internalization (recognition assay), alveolar macrophages (1 × 10
5) were seeded onto the glass insert of MatTek 35 mm dishes and incubated for 2 h [
12]. Cells were washed then incubated with GFP
-C. albicans in phenol red-free DMEM containing penicillin, streptomycin and 0.1 % endotoxin-free BSA (stimulation media) for 30 min at 37 °C and 5 % CO
2. Macrophages were washed, fixed with 4 % paraformaldehyde for 15 min and then stained with DAPI. Images were captured on a Marianas 200 spinning disk confocal microscope using Intelligent Imaging Innovation Inc. (3I) software (Slidebook 6.0) to determine the number of macrophages containing GFP-
C albicans. For killing assays, alveolar macrophages (in 48 well plates) were incubated for 2 h in stimulation media with GFP-
C. albicans. Wells containing an equivalent number of GFP-
C. albicans (without macrophages) were included as a positive control for determining 100 % viability. Macrophages were lysed with 1 % Triton X-100 and GFP-
C. albicans viability was measured using the XTT Cell Viability Kit as described [
32].
Bone marrow neutrophils were isolated from untreated cPLA
2α
+/+ and cPLA
2α
−/− mice as described previously and purity (>95 %) determined on cytospins [
33]. Neutrophils (1 × 10
5) were plated on polylysine-coated MatTek 35 mm dishes, incubated for 1 h and then incubated with GFP
-C. albicans for 30 min. After fixation the cells were incubated for 1 h in PBS containing 10 % FBS and then incubated overnight with anti-Ly6G antibody followed by treatment with anti-rabbit AF594 secondary antibody and with DAPI. For killing assays, GFP-
C. albicans was added to neutrophils (5 × 10
4) in the 96 well plates, centrifuged for 5 min at 300 g to synchronize the infection, and then incubated for 2 h at 37 °C and 5 % CO
2. GFP-
C. albicans viability was determined as described above for macrophages.
Statistics
The data are presented as mean ± SEM and analyzed using the 2-tailed unpaired t-test or the Mann Whitney method to determine statistical significance (defined as p < 0.05).
Discussion
cPLA
2α is a highly conserved enzyme that is widely expressed throughout all tissues in mice and humans, and is rapidly activated by diverse agonists through common signaling pathways [
1]. It is the only mammalian PLA
2 that preferentially releases
sn-2 arachidonic acid from phospholipids and its role in initiating the production of eicosanoids is well documented [
40,
41]. Identification of humans with cPLA
2α deficiency has confirmed that it mediates eicosanoid production and functions in homeostatic processes important for human health [
42‐
45]. cPLA
2α has been implicated in regulating both normal physiological processes and disease pathogenesis in many organ systems from studies using cPLA
2α
−/− mice, however, the specific mechanisms involved in many cases have not been elucidated [
1,
46,
47]. In models of lung disease, cPLA
2α
−/− mice are protected from pulmonary fibrosis, acute lung injury and allergic responses [
48‐
50]. Since lung fibrosis and allergic lung responses are exacerbated in COX-1
−/− and COX-2
−/− mice but reduced in 5-LO
−/− mice, the results suggest that in certain pro-inflammatory disease states cPLA
2α contributes to disease through a dominant role for pro-inflammatory leukotrienes [
51‐
54]. By comparing cPLA
2α
+/+ and cPLA
2α
−/− mice in this study, we are probing the primary mechanism for eicosanoid production in vivo in response to exposure of the lung to the opportunistic pathogen
C. albicans. This model reflects the collective influence of lipid mediators resulting from cPLA
2α activation in regulating innate immune responses. Immune competent mice are resistant to infection from intratracheal instillation of
C. albicans, which is rapidly cleared from the lungs with minimal health effects due to contributions from both alveolar macrophages and neutrophils in host defense [
35]. Our results suggest that cPLA
2α contributes to innate immune defense mechanisms in the lung to control
C. albicans infection and dampen inflammation.
cPLA
2α
−/− mice do not clear
C. albicans from the lung as efficiently as cPLA
2α
+/+ mice and exhibit greater signs of inflammation including excessive weight loss, increased production of pro-inflammatory cytokines and increased neutrophil recruitment to the lung. Pro-inflammatory cytokines (TNFα, IL1α, IL1β) are higher in cPLA
2α
−/− than cPLA
2α
+/+ mice 6–24 h after
C. albicans infection. In mouse models of bacterial pneumonia these cytokines are produced by alveolar macrophages from initial interaction with pathogens and signal to epithelial cells and neutrophils to mount responses to infection [
55‐
57]. Alveolar macrophages, isolated 6 h after intratracheal instillation, contain engulfed GFP-
C. albicans indicating that the fungi reach the alveoli shortly after instillation. Pro-inflammatory cytokines have been shown to induce the production of neutrophilic chemokines such CXCL1, which is higher in cPLA
2α
−/− mice and correlates with the elevated neutrophil influx [
56,
58].
C. albicans infection in cPLA
2α
+/+ mice leads to a small but significant increase in production of TNFα, IL1α and IL1β, and induces neutrophil influx, although at lower levels than in cPLA
2α
−/− mice. It is likely that these innate immune responses in cPLA
2α
+/+ mice are important for host defense resulting in clearance of
C. albicans from the lung. It has been shown that TNFα, IL1α and IL1β are important for host defense against invasive
C. albicans infection in mice [
59,
60]. However, the exaggerated responses to
C. albicans infection in cPLA
2α
−/− mice point to an important role for cPLA
2α in regulating the balance of cytokines produced for effective microbial clearance without excess inflammation that may cause tissue injury and dissemination of
C. albicans from the lung. This may in part be due to higher levels of PGE
2 in cPLA
2α
+/+ mice since prostaglandins suppress the production of TNFα, IL1α and IL1β [
15,
61‐
63]. PGE
2 is also important in maintaining endothelial barrier function, promoting wound healing and inhibiting neutrophil migration [
64]. PGI
2 also has anti-inflammatory properties [
3]. Our results show relatively high levels of endogenous PGI
2 in BALF suggesting constitutive production perhaps by vascular endothelial cells and smooth muscle cells reflecting its important role in maintenance of the vasculature [
65]. PGI
2 levels were similar in BALF from cPLA
2α
+/+ and cPLA
2α
−/− mice, and not increased by
C. albicans infection, suggesting another PLA
2 is involved in its production and that it is not involved in the phenotypic differences observed during
C. albicans infection.
Of the cytokines measured in BALF, IL6 showed the greatest increase in cPLA
2α
−/− mice early after
C. albicans instillation reaching levels 10-fold higher than in cPLA
2α
+/+ mice. IL6 is an indicator of disease severity, reflecting the more pronounced effect of
C. albicans on the health of cPLA
2α
−/− compared to cPLA
2α
+/+ mice, which show only a small increase in IL6 production [
66]. IL6 is considered a pleiotropic cytokine made by immune and stromal cells in response to diverse agonists that has a homeostatic function and regulates immunity [
67]. IL6 regulates the recruitment of leukocytes during infection and may contribute to the higher neutrophil influx in cPLA
2α
−/− mice [
67,
68]. Although IL6 can be induced by prostaglandins, its higher level in cPLA
2α
−/− mice suggests that it is directly made by cells in response to
C. albicans perhaps through the early production of TNFα, IL1α, and IL1β [
66,
69,
70]. In contrast to the results of this study, cPLA
2α
−/− mice are protected during
Pseudomonas aeruginosa lung infection that correlates with decreased IL6 production [
71]. Therefore, cPLA
2α can exacerbate infection or have a protective role in the lung depending on the type of pathogen.
Leukotrienes also regulate immunity in the lung during infection by promoting trafficking of neutrophils, T lymphocytes, dendritic cells and vascular permeability [
2,
7]. Mice deficient in leukotriene production are more susceptible to bacterial (
Klebsiella pneumonia, Mycobacterium tuberculosis) and fungal (Histoplasmosis) lung infection showing impaired microbial clearance and survival [
72‐
74]. However there are differences in the responses of leukotriene-deficient mice to bacterial and fungal infection. Following bacterial challenge, 5-LO
−/− mice have reduced neutrophil influx in the lung [
72]. However,
Histoplama capsulatum lung infection in 5-LO
−/− mice results in increased neutrophil recruitment and greater production of pro-inflammatory cytokines than in wild type mice, as we observed in
C. albicans-infected cPLA
2α
−/− mice. Leukotrienes regulate innate immune responses in part by enhancing alveolar macrophage phagocytosis and microbial killing [
72,
74].
Our results demonstrate that alveolar macrophages and neutrophils from uninfected cPLA
2α
−/− mice have a reduced capacity to kill
C. albicans than cells from cPLA
2α
+/+ mice. We previously reported that
C. albicans poorly activates cPLA
2α in alveolar macrophages from cPLA
2α
+/+ mice and induces very little eicosanoid production, although it is enhanced by priming with GM-CSF due to increased expression of dectin-1 [
12]. Therefore it is not likely that this inherent difference in the killing capacity of alveolar macrophages from uninfected cPLA
2α
+/+ and cPLA
2α
−/− mice is due to production of endogenous eicosanoids during the killing assay in vitro. The basis for this inherent difference in
C. albicans killing is not known but the lack of eicosanoids during development of cPLA
2α
−/− mice may affect gene expression that influences killing of
C. albicans. The results also showed that alveolar macrophages isolated from cPLA
2α
−/− mice 6 h after instillation of GFP-
C. albicans have fewer engulfed GFP-
C. albicans than macrophages from cPLA
2α
+/+ mice. It is likely that cells are primed by cytokines in vivo to enhance production of eicosanoids and regulate killing of
C. albicans.
A role for the epithelium during
C. albicans lung infection is suggested by results showing that cPLA
2α
−/− mice have higher levels of CCL20 and CSF2 than cPLA
2α
+/+ mice. During lung infection CCL20 and CSF2 (GM-CSF) are derived from lung epithelium and contribute to recruitment of dendritic cells and neutrophils [
55,
58,
75]. The lung epithelium may also contribute to production of pro-inflammatory cytokines since
C. albicans stimulates oral and vaginal epithelial cells to produce chemokines and cytokines including IL1α, IL1β and TNFα [
76,
77]. Although this has not been investigated in lung epithelial cells, there may be a local immune response at the lung mucosa for combating
C. albicans in cPLA
2α
+/+ mice. It is interesting that
C. albicans disseminates to the kidney in cPLA
2α
−/− mice suggesting there is damage to the epithelial/endothelial barrier possibly due to the increased inflammation. Since alveolar epithelium damage can be sensed by alveolar macrophages this may lead to heightened pro-inflammatory responses as we observed in cPLA
2α
−/− mice [
78]. The results suggest that cPLA
2α activation is an important mechanism for regulating the function of immune and stromal cells in the lung to protect from
C. albicans infection.