Increased susceptibility to pulmonary Pseudomonas infection in Splunc1 knockout mice

Splunc1 knockout mice and animal husbandry

Splunc1 null mice were generated through screening of an ENU mutagenized sperm archive as previously described (14). The homozygous Splunc1^+/+ mice and Splunc1^−/−mice were bred to maintain their homozygosity and to generate a mouse cohort of both WT and Splunc1 KO mice with the same genetic background for the experiments. Mice were maintained in a specific pathogen-free status in a 12-h light/dark cycle. All procedures were conducted using mice 6–9 weeks of age maintained in ventilated microisolator cages housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facility. Protocols and studies involving animals were conducted in accordance with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

Real-time PCR analysis

Total mRNA was isolated from the upper two lobes of right lung tissues of WT and Splunc1 KO mice using Trizol reagent (Life Technologies, Carlsbad, CA). Quantitative PCR (qPCR) was performed using ABI7900HT (Applied Biosystems, Foster City, CA) and primers of Muc5ac, Muc5b, CCSP, Foxj1, Cxcl1, Cxcl2, Cxcl5, Ccl20, LL37, Lactoferrin, Lysozyme, and β-Defensin 2. Validation tests were performed to confirm equivalent PCR efficiencies for the target genes. Test and calibrator lung RNAs were reverse transcribed using a High-Capacity cDNA reverse transcription kit (Life Technologies), and PCR was amplified as follows: 50°C for 2min, 95°C for 10min, 40 cycles; 95°C for 15s; 60°C for 1min. Three replicates were used to calculate the average cycle threshold for the transcript of interest and for a transcript for normalization (β-glucuronidase [GUS-B]; Assays on Demand; Applied Biosystems). Relative mRNA abundance was calculated using the ΔΔ cycle threshold (Ct) method.

Gram-negative bacteria Pseudomonas aeruginosa

The Pseudomonas aeruginosa strain (PAO1, ATCC BAA-47) was used for all experiments. PAO1 obtained from a single colony was stored in aliquots at −80°C in 15% glycerol/ tryptic soy broth (TSB). For each experiment, an aliquot of bacteria was grown for 16h at 37°C in autoclaved TSB with shaking. An aliquot of the overnight grown bacteria was then diluted 1ml into 5ml fresh TSB and incubated for an additional 2h at 37°C with shaking. Bacteria were washed twice and resuspended in 10 ml phosphate-buffered saline (PBS).

In vivo exposure of mice to Pseudomonas aeruginosa

Mice were anesthetized with inhalation of isoflurane and treated with PAO1 through intranasal instillation of ~1.5 × 10⁷ CFU per mouse in 30μl PBS. Control mice were intranasally inoculated with 30ul of PBS. For the survival test, mice were anesthetized with 2.5% avertin and then infected with an in intranasal instillation of PAO1 at a dosage of 10⁹ CFU/mice.

CFU assay

The number of CFU was determined by serial dilution and quantitative culture on TSB agar plates. The left lung lobe was homogenized in 1ml saline and placed on ice. Dilution of 100μl of lung tissue homogenate or bronchoalveolar lavage fluid (BALF) was mixed with 900μl saline. Four serial 10-fold dilutions in saline were prepared and plated on TSB agar plates and incubated for 18h at 37°C, each dilution plated in triplicate. The colonies were then counted and surviving bacteria were expressed in log₁₀ units.

BALF and cell differential counts

At 6h or 24h after treatment of PAO1, mice (6 mice/ group) were anesthetized with 2.5% tribromoethanol (Avertin). The trachea was cannulated, the lungs were lavaged twice using 1ml saline, and the BALF samples pooled. A 16μl aliquot was stained with 4μl Acridine orange (MP Biomedical, Santa Ana, CA), and cells were counted with a Vision Cell Analyzer cell counter (Nexcelom, Lawrence, MA). An additional aliquot was placed onto glass microscope slides (Shanon Cytospin; Thermo Fisher, Pittsburgh, PA), stained with Diff-Quick; cell differential was determined microscopically. A total of 400 cells of every slide were counted at least twice for inflammatory cell differential counts.

Cytokine assay

Cytokine levels in BAL were quantified using the mouse Cytokine Multiplex Panel Milliplex assay (Millipore, Billerica, MA). The expressions of IL-1β, IL-6, IL-10, IL-12(p70), IL-17, IFN-γ, TNF-α, GM-CSF, KC, IP-10, VEGF and MIP-1α were analyzed using the Luminex assay system, based on manufacturer's instructions. Standard recombinant protein solution was used to generate a standard curve for each analyzed protein. Absolute cytokine concentrations were calculated from the standard curve for each cytokine.

Biofilm assay

A slightly modified version of the microtiter plate assay developed by O'Toole and Kolter was used (15). Briefly, overnight planktonic cultures of PAO1 were inoculated into 100 μL of DMEM in a 96-well culture-treated polystyrene microtiter plate (Fisher Scientific, Pittsburgh, PA) with or without recombinant SPLUNC1 or antibiotic controls. Wells filled with growth medium alone were included as negative controls. After 3 hour incubation at 37°C, surface-adherent biofilm formation was measured by staining bound cells for 15 minutes with a 0.5% (w/v) aqueous solution of crystal violet. After rinsing with distilled water, the bound dye was released from the stained cells using 95% ethanol, and OD was determined at 590 nm.

Static bacteria-epithelial cells co-culture biofilm assay

Primary mouse tracheal epithelial cell cultures were established as previously described (14). Briefly, Splunc1^−/− mice and Splunc1^+/+ mice were euthanized and their tracheas immediately removed. Tracheal epithelial cells were dissociated and seeded onto collagen-coated, semi-permeable membranes with a 0.4-μm pore size (Millicell-HA; surface area, 0.6 cm²; Millipore Corp., Bedford, MA). Cells were maintained in small airway epithelial cell growth medium (Lonza, Allendale, NJ) and the mucosal medium was removed 24 hours after seeding and the cells were allowed to grow and differentiate at the air-liquid interface.

To assess the viability of bacteria, biofilms were grown on polarized and confluent mouse tracheal cells under static conditions as previously described (16). Briefly, PAO1 were inoculated on the apical surface of epithelial cells grown on filters (MOI=25). After 1 hour of incubation at 37°C, unattached bacteria were removed. Filters containing epithelial cells and the attached bacteria were returned to 37°C and 5% CO₂ for the duration of each experiment (5 hours). Arginine was added to the medium in order to prolong the viability of tracheal epithelial cells incubated with bacteria under static conditions. At the end of the treatment, biofilms remaining at the apical side of airway cells were washed once with Minimal Earle's medium, and then 0.1% Triton X-100 was added to the medium for 15 minutes to lyse the epithelial cells and dissociate the biofilms. The lysate was vortexed for 3 minutes and serial dilutions were spot titered onto LB plates to determine the colony-forming units (CFU)/ml.

Lung histopathology

Lung tissues were harvested at 6h and 24h after infection, inflation fixed in situ with 4% paraformaldehyde at 10cm H₂O for 10 minutes with the chest cavity open. The right lobe was embedded in paraffin and 5μm sections were prepared. Sections were stained with hematoxylin and eosin, and histological evaluation was performed to examine bacterial infection-induced pathological severity. The stained lung sections were evaluated in a double-blind fashion under a light microscope, using a histopathologic inflammatory scoring system as described previously (17).

Data analysis

Data are expressed as mean ± SEM. Statistical comparisons between the groups of mice were made using ANOVA, followed by Dunnett's multiple comparison test (one way ANOVA) or the Bonferroni multiple comparison test (two way ANOVA). A p value <0.05 was considered to be statistically significant. Determinations of the significance of the differences in survival outcomes between Splunc1 KO mice and WT control littermates after PAO1 infection were calculated by Kaplan–Meier survival curve comparisons, as well as the p values derived from a log-rank test.

Splunc1 is critical for pulmonary host defense against Pseudomonas aeruginosa in vivo

To determine if Splunc1 affects susceptibility to PAO1-induced lung infection, groups of 6- to 9-week-old Splunc1 WT and KO mice were challenged through intranasal instillation. We carried out preliminary studies to determine the optimal dosage for survival experiments. There was no mortality within 48h in WT mice when concentrations of PAO1 at 10⁷ and 10⁸ CFU/animal were administered. We determined the survival of mice against PAO1 infection at a dosage of 10⁹ CFU/animal to reflect the severity of acute respiratory infection. When both Splunc1 WT and KO mice were challenged with 10⁹ CFU/animal of PAO1, KO mice showed accelerated and increased mortality (log-rank test, p = 0.0056) (Fig.1). Upon PAO1 bacterial infection, Splunc1 WT mice started showing mortality at 18h post infection, while other infected mice gradually died throughout the time period until all mice died at 52h after infection (mean survival time 29.53 ±11.79h). The death of Splunc1 KO mice was observed as early as 10h after bacterial infection, whereas all the remaining Splunc1 KO mice succumbed to PAO1-induced lung infection within 44h (mean survival time 20.21 ± 8.00h). These results indicate that Splunc1 is essential to prolonged survival following PAO1 infection.

Splunc1 deficiency is associated with increased bacterial burdens in the lung

In order to delineate the underlying mechanisms for Splunc1-regulated susceptibility to PAO1 infection, the different host innate immune responses of Splunc1WT and KO mice were first examined. To determine the bacterial burden in lungs of Splunc1 KO and WT mice, animals were intranasally challenged with 1.5 ×10⁷ CFU/mouse of PAO1 and sacrificed at 6h and 24h after PAO1 infection. The ablation of Splunc1 expression resulted in increased sensitivity to bacterial infection. Splunc1 KO mice showed a 2.5-fold higher level of PAO1 in the left lung when compared with the WT control mice at 6h and 24h post-infection (Fig. 2A). Six hours after PAO1 exposure, about 10-15% of lung bacterial burden could be detected in the BAL; Splunc1 KO mice showed a non-significant tendency towards increased levels of bacteria, compared to those of WT mice (Fig.2B). However, at 24h after infection, the bacterial burden in the BAL was significantly higher in Splunc1 KO mice than in WT mice. These data suggest that deficiency of Splunc1 results in increased susceptibility to bacterial infection.

Splunc1 deficiency results in increased Pseudomonas aeruginosa–induced inflammation

To determine if the increased lung bacterial burden in Splunc1 KO mice results in increased inflammation, we analyzed the BAL cell profile and lung tissue inflammation in the lungs of Splunc1 KO and WT mice at 6h and 24h following PAO1 infection (Fig. 3). BAL total cell count (2~4 × 10⁵cells/lung) or differentials (≥98% macrophages) from unexposed Splunc1 KO mice were not different than those from WT control mice. After 6h of bacterial exposure, the total inflammatory cell counts in BALF increased in both Splunc1 KO and WT mice, but the increase was greater in Splunc1 KO mice than the increase in WT mice. The difference in increased total inflammatory cell number between the Splunc1 WT (71.06±16.25 × 10⁵/ml) and KO (126.08±18.72 × 10⁵/ml) mice was statistically significant. The total macrophage number in the BAL at 6h after infection was higher for Splunc1 WT mice (28.33±6.21 × 10⁵) than it was for KO mice (16.63±3.20 × 10⁵), which may suggest more effective bacterial killing in WT mice. Conversely, the total polymorphonuclear (PMN) cell number was significantly lower in Splunc1 WT control mice (51.45±18.24 × 10⁵) than in KO mice (109.22±20.25 × 10⁵) (Figs.3B-C). The total BAL cell count decreased at 24h after exposure in both Splunc1 WT and KO mice, but there were still more inflammatory cells in the KO mouse group. PAO1 infection resulted in increased interstitial inflammation in the lungs (Fig. 4). We observed the increased number of inflammatory cells after Pseudomonas infection in both airways and alveolar regions, but the increase was more prominent in the alveolar region. To determine whether PAO1 infection-induced lung inflammation was influenced by Splunc1, lung histopathology was assessed after infection. Lungs from uninfected WT control mice were not different than those from Splunc1 KO mice (data not shown). There was slightly more infiltration of PMNs in Splunc1 KO mice than in their WT littermates at both 6h and 24h after infection (Fig 4). Moreover, lung tissues from Splunc1 KO mice (Figs 4B&4D) contained more areas of focal peribronchial and alveolar neutrophilic infiltrates than did lungs from WT control mice (Figs 4A&4C).

Elevated pro-inflammatory cytokine and chemokines production in Splunc1 KO mice following PAO1 challenge

Because Splunc1 has been suggested to possess anti-inflammatory functions (7), we analyzed the cytokine profile of the lungs of Splunc1 KO and WT mice after bacterial infection. Without PAO1 exposure, BALF cytokine levels were not different between Splunc1 KO and WT mice. These pro-inflammatory cytokines were undetectable in naïve mice (data not shown). After exposure, the spectrum of BALF cytokines that were changed did not vary by genotype, but the magnitude of the response was consistently higher in the BALF from Splunc1 KO mice, compared with that of BALF from WT mice (Fig.5). Pro-inflammatory cytokines including IL-1β (Fig.5A) and chemotactic cytokines, chemokine (C-X-C motif) ligand 1(Cxcl1, also known as KC; Fig.5B), were significantly increased in BALF from Splunc1 KO mice as compared with that from WT mice. At 6h after PAO1 infection, chemokine (C-C motif) ligand 3 (Ccl3, also known as MIP-1α; Fig.5C) increased significantly in BALF from Splunc1 KO mice. Expression of IL-6 in BALF was considerably higher in Splunc1 KO mice than in WT mice at 24h post-inoculation (Fig.5D).

Both BAL cell counts and histological images suggested that there was a more significant increase of PMNs in Splunc1 KO mice than in WT mice; we further examined the expression of several chemokines in order to distinguish their contribution to the increased PMNs after PAO1 infection. Without PAO1 exposure, the expression of Cxcl1 was slightly less in Splunc1 KO mice, while Cxcl2, Cxcl5, and Ccl20 were expressed similarly in Splunc1 KO and WT mice. The expression of Cxcl1 (4-fold), Cxcl2 (2-fold), and Ccl20 (4-fold) were all significantly elevated in Splunc1 KO mice when compared to the expression in WT mice at the 6h time point after PAO1 infection (Fig.6A-C). This tendency was preserved at the 24h time point, when Splunc1-deficient mice maintained a 2-fold level of elevated Cxcl1 and Cxcl2 levels as compared to levels of the WT mice (Fig.6A-B). Interestingly, there was no difference between Splunc1-deficient mice and their WT littermate mice in regards to the expression of Cxcl5, also known as epithelial-derived neutrophil-activating peptide 78 (ENA-78), either before or after the PAO1 challenge (Fig.6D).

Splunc1 deficiency changes epithelial secretory properties

Secretory proteins from airway epithelium, such as mucin and Clara cell secretory protein (CCSP), provide components of extracellular lining fluid in the airways and play a major role in mucociliary clearance. Since Splunc1 is one of the most abundant secretory proteins in the airway, and Splunc1-expressing cells overlap with both mucus- and CCSP-producing cells in mouse airways (data not shown), we determined if the ablation of Splunc1 in mouse airway secretory cells results in defects in other major secretory proteins. Interestingly, the expression of two major airway mucin genes, Muc5ac and Muc5b, as well as CCSP, were all significantly decreased in naïve Splunc1 KO mice than WT littermates at baseline, prior to any bacterial challenge (Fig.7A-C). The decreased expression of Muc5ac, Muc5b, and CCSP in Splunc1 KO mice persisted after the PAO1 challenge at both 6h and 24h post-infection (Fig.7A-C). Forkhead box protein J1 (Foxj1), encoded by the Foxj1 gene, is involved in ciliogenesis and a marker of ciliated cells in airway epithelium. Although Splunc1 is only expressesed in secretory cells of airway epithelium, the deficiency of Splunc1 also results in decreased expression of Foxj1 (Fig.7D). Our data suggest that the secretory properties of airway epithelium are likely disrupted when Splunc1 is ablated.

Effects of Splunc1 ablation on pulmonary antimicrobial gene expression

To further understand how Splunc1 deficiency and the decreased secretion in Splunc1 KO mice influence host defense after pulmonary infection, we examined the expression of several major antimicrobial molecules. The expression of cathelicidin antimicrobial peptide (CAMP, also known as LL37, FALL39, or CAP18) and lactoferrin was slightly decreased prior to bacterial infection in Splunc1 KO mice, while the expression of lysozyme, one of the most abundant antimicrobial proteins, did not vary between Splunc1 KO and WT mice before infection (Fig.8A-C). Six hours after PAO1 infection in WT mice, the magnitude of the increased expression of LL37 was highest (271-fold, p=0.0376), followed by the expression of lactoferrin (20-fold, p=0.0093), while there was only a 60% increase in the expression of lysozyme (Fig.8A-C), when compared with PBS control. At 24h after PAO1 infection, the expression of LL37 remained 24-fold higher than PBS control (Fig.8A, p=0.1814), while only a minimal increase was observed in expression of both lactoferrin and lysozyme (Fig.8B-C). However, the inductions of LL37, lactoferrin, and lysozyme in the lungs from Splunc1 KO mice were all significantly suppressed compared with those of WT mice at both 6h and 24h after PAO1 infection. Surprisingly, the expression of P-defensin 2 did not fluctuate much in mouse lungs with or without PAO1 infection, and there was no difference in the expression of β-defensin 2 between Splunc1 KO and WT mice (Fig.8D).

SPLUNC1 is essential in preventing biofilm formation by Gram-negative bacteria

To determine if Splunc1-deficiency associated changes in epithelial secretion and antimicrobial molecules affect P. aeruginosa biofilm formation, we co-cultured PAO1 on the apical surface of polarized mouse primary airway epithelial cells. PAO1 forms surface-associated bacterial biofilms on polarized mouse primary airway epithelial cells when co-cultured for six hours at 37°C, as measured by colony forming units. The ablation of Splunc1 in KO mice significantly increased PAO1 biofilm formation (Fig 9A) when comparing with co-cultured epithelial cells from WT mice.

To investigate whether exogenously addition of SPLUNC1 can conversely prevent PAO1 biofilm formation, we carried out microtiter plate based biofilm assays. We found that, when PAO1 was incubated with recombinant SPLUNC1 (rSPLUNC1) at increasing concentrations, biofilm biomass decreased and was inversely related to the rSPLUNC1 concentration (Fig 9B).