Acute intratracheal Pseudomonas aeruginosa infection in cystic fibrosis mice is age-independent

Infection experiments were performed with two CF mouse models (a) B6.129P2(CF/3)-Cftr ^{TgH(neoim)Hgu} , (b) Cftr ^tm1Unc -Tg(FABPCFTR)1Jaw/J and their respective littermates. According to the nomenclature of Teichgräber et al. the mouse lines are called (a) Cftr ^MHH and (b) Cftr ^KO , their non-CF littermates (a) B6 and (b) WT, respectively. In Cftr ^{TgH(neoim)Hgu} mice the exon 10 of the Cftr gene had been disrupted by the insertion of the vector pMCIneoPolyA [16]. Since those mice produced low levels of Cftr [17] but showed a mixed genetic background [24], from the original Cftr ^{TgH(neoim)Hgu} mutant mouse, CF strain CF/3-Cftr ^{TgH(neoim)Hgu} was established at the Institute of Laboratory Animal Science of the Hannover Medical School by brother-sister mating for more than 40 generations. Next, the congenic mouse inbred strain B6.129P2(CF/3)- Cftr ^{TgH(neoim)Hgu} , which is used in this study, was generated by 40 backcross generations using CF/3-Cftr ^{TgH(neoim)Hgu} as donor strain and C57BL/6J as recipient strain [25]. Following the nomenclature of Teichgräber et al. [19] this strain is called Cftr ^MHH , syngenic C57BL6/J mice are called B6 and served as controls. Cftr ^MHH mice are regulary monitored for their congenic C57BL/6J status using 27 SNP markers and integrity of the mutant Cftr locus by intragenic microsatellite markers [24, 26, 27].

STOCK Cftr ^tm1Unc -Tg(FABPCFTR)1Jaw/J mice were obtained from the Jackson Laboratories. These mice, in the following called Cftr ^KO , which are of a mixed genetic background consisting of C57BL/6, FVB/N and 129, are knock-outs for the murine Cftr gene, but express human CFTR in the gut under control of the FABP1 (fatty acid binding protein1) promoter, which prevents acute intestinal obstruction 1 [12, 18]. Mice were obtained homozygous and heterozygous for the Cftr ^tm1Unc targeted mutation (tm/tm and tm/+) as well as homozygous and hemizygous for the FABP-hCFTR transgene (tg/tg and tg/0). Tm/+ tg/0 mice were used as parents to generate wildtype control mice (called WT). Genotyping was performed using the protocols provided by the JAX lab [28].

Mice were maintained at the Central Animal Facility of the Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. They were held in groups of three to five animals animals in microisolator cages (910 cm²) with filter top lids and free access to sterilised standard laboratory chow (diet No. 1324, Altromin, Lippe, Germany) and autoclaved, acidulated water at 21 ± 2°C, 55 ± 5% humidity and a 10:14 light-dark-cycle. None of the CF mice showed gastrointestinal complications which would require a special diet. All mice were regularly monitored for infection by typical pathogens according to the FELASA recommendations [29]. All procedures performed on mice were approved by the local district governments (AZ. 33.9-42502-04-08/1528) and carried out according to the ILAR guidelines for the care and use of laboratory animals [30]. Experimental groups of mice were allocated by age of either younger than three (henceforth designated as young) or elder than six months (henceforth designated as old). This selection aimed to mimic the categories of Teichgräber et al. [19] that mice older than 4 months were susceptible to an infection with P. aeruginosa whereas the younger mice were more or less resistant.

Due to breeding limitations B6 mice showed a strong predominance of males and no 192 h value of old B6 exist.

Non-invasive head-out spirometry with 14 parameters was performed on conscious restrained mice [22]. In brief, four mice were investigated in parallel by placing them in glass inserts with their heads protruding out through a set of membranes ensuring an airtight fit. Respiration caused air to flow through a pneumotachograph positioned above the thorax of the animals. The airflow was digitalized and analyzed with the Notocord Hem 4.2.0.241 software package (Notocord Systems SAS, Croissy Sur Seine, France).

Pseudomonas aeruginosa strain TBCF10839 [31] was grown in Luria broth (LB) overnight at 37°C. The overnight culture was washed twice with the same volume of sterile PBS to remove cell detritus and secreted exopolysaccharides, then the optical density of the bacterial suspension was determined and the intended number of colony forming units (CFU) was extrapolated from a standard growth curve. Inocula with 6.0 × 10⁵ CFU in 30 μl were prepared by dilution with sterile PBS. This infection dose is approximately one tenth of the LD50 of strain TBCF10839 for C57BL/6J mice and was able to produce a clinical infection without mortality.

Mice were infected intratracheally (i.t.) with 6.0 × 10⁵ CFU of P. aeruginosa strain TBCF10839 under a light anaesthesia. For detailed description of the view-controlled intratracheal instillation see Munder et al. [23]. To characterize the course of the bacterial infection, the body condition, weight, rectal temperature and lung function of the mice were evaluated as described previously [32]. In brief the overall health of the animals was assessed by vocalisation, piloerection, posture, locomotion, breathing, curiosity, nasal secretion, grooming and dehydration. Dysfunctions in single parameters were assessed by zero, one or two points. The overall fitness of the mice was determined by adding the points resulting in the following score of the mouse body condition: unaffected (0-1); slightly affected (2-4); moderately affected (5-7); severely affected (8-10); moribund (≥ 11).

Non-invasive head-out spirometry. First, spirometric values of uninfected animals (B6, WT, Cftr ^KO and Cftr ^MHH with age young: < 99 days and old: > 179 days) were averaged (median) from three independent measurements preformed on consecutive days. Prior measurements assured that the mice had adapted to the procedure. Lung function measurements of infection experiments were taken daily at the five days prior to inoculation and at time points 4, 6, 8, 10, 12, 18, 24, 48, 72, 96, 120, 144, 168, 192 hours post inoculation.

Forty-eight hours after challenge subgroups of mice were euthanized. Their left lungs were taken for the determination of bacterial counts and the right lungs were stained and examined histologically.

The right lungs were fixed via the trachea (4% paraformaldehyd), embedded in paraffin and stained with haematoxilin/eosin. One section was selected that showed aspects from all three lobes of the right lung. This slide was examined in twenty fields of view at a 100 fold magnification using a Zeiss Axiophot photomicroscope. Inflammation was assessed using a semi quantitative pathohistological score [22]. Shortly, lung histological changes were scored on a scale from one to three points. Points were given separately for lung parenchyma, airways and lung vessels. The total score classified airway inflammation into the categories: almost not visible (0-5); slight (6-20) moderate (21-40); severe/profound inflammation (41-60). In the current study no more than a medium-grade inflammation was seen, appearing as a purulent alveolar pneumonia with peribronchiolar and perivascular inflammatory infiltrates.

The left lungs of the euthanized mice were ligated, resected and homogenised. Aliquots were plated and bacterial numbers of whole organs were calculated. Previous experiments showed that the distribution of bacteria is approximately equal in left and right lungs after i.t. application (data not shown).

Each CF mouse model and its wild type controls were investigated separately by age group using non-parametric test statistics of SPSS 16 (Version 16.0.2, SPSS Inc, Chicago, USA). p-values (p < 0.05) with subsequent Bonferroni correction were calculated by 2-sided Monte Carlo simulations (100,000 simulations). Hereby groups were composed of equal numbers of mice (perfect match approach).

To evaluate the impact of age, genetic background and the Cftr mutant construct on respiratory health, we determined lung function in young and old CF mice and their congenic wild-type littermates (Figure 1).

Tidal volume increased with age of the animals reflecting an increase in body mass. Respiration decreased, also characterized by increasing times for one breath. The slower breathing was also characterized by a decrease in the flows of expiration and inspiration.

Body weight measurements showed that Cftr ^KO and WT mice were slightly larger and heavier than Cftr ^MHH and B6 mice. This is mirrored in a slightly higher tidal volume for the Cftr ^KO and WT mice.

All mice irrespective of genotype or gender had a comparable total lung volume (tidal volume, Figure 1A). CF mice, however, achieved the comparable lung volume through an increase in respiratory rate (Figure 1B). Correspondingly the time for one breath (Time of inspiration plus expiration, Figure 1C) was smaller in CF mice. The higher respiratory rate was associated with higher flows as depicted by the EF50 parameter (Midtidal expiratory flow at 50% expiration, Figure 1D).

To monitor the outcome of an intratracheal instillation of P. aeruginosa in our CF mouse models and their non-CF littermates, mice of both genders were exactly matched with their corresponding wild type controls by sex, age and body weight. Mice were characterized in their global health score (Figure 2), rectal temperature (Figure 3), body weight (Figure 4) and lung function (Figure 5, Additional file 1) at 14 time points over a period of 192 h.

B6 mice were more affected by P. aeruginosa in their body condition than the Cftr ^KO and their non-CF littermates (Figure 2). With the exception of young Cftr ^KO mice the corresponding CF and non-CF mouse lines exhibited a similar time course of disease symptoms. Animals were notably affected between 6-12 h after inoculation and recovered within the next 48 h. Young WT mice were least affected by the instillation of P. aeruginosa into their lungs.

Upon exposure to bacteria mice did not react with fever but with a drop of body temperature. The temperature profile mimicked the time course of the global health score. Cftr ^MHH and the B6 mice experienced a stronger drop of temperature than the Cftr ^KO and WT mice, the latter being minimally affected with a maximal reduction of rectal temperature of 5°C (Figure 3).

Within the first 24 h post inoculation the mice lost 8% (old B6) to 13% (young Cftr ^MHH ) of their initial body weight (Figure 4). By the end of the experiment the young mice had almost completely regained their initial weight, whereas an irreversible weight loss was observed in all old mice. Consistent with an intestinal CF phenotype, old Cftr ^MHH mice were significantly lighter than their congenic B6 mice [24].

The time course during infection is shown exemplarily for tidal volume (the total volume inspired and expired in one breath) (Figure 5A, B), Time of inspiration plus expiration (Figure 5C, D) and EF50 (Figure 5E, F). The data of all 14 measured lung function parameters are shown in the online supplement (Additional file 1). The old non-CF and CF mice showed a similar response in lung function towards the instillation of P. aeruginosa into their lungs. Young KO mice differed from their WT FABP littermates in a shorter 'time of pause' prior to and after inoculation, but not in any other of the 14 parameters. In contrast, non-infected young Cftr ^MHH mice showed another breathing pattern than their congenic B6 mice (see above). Cftr ^MHH mice differed from their non-CF congenics in seven lung function parameters with a high respiratory rate as the leading symptom. This characteristic pattern of non-infected Cftr ^MHH mice was also seen in the challenged mice at the late time points of 144, 168 and 192 h when they had recovered from infection. Thus, by the end of experiment the Cftr ^MHH mice had regained the initial lung function phenotype. Besides these CF-genotype-driven differences between congenic mice that were apparently independent of bacterial infection, a differential response of Cftr ^MHH and B6 mice was noted at time points 4 and 6 hours after challenge with P. aeruginosa. Lung function slightly, but significantly differed in seven flow or volume parameters (Additional file 1). In summary, differences in lung function between congenic non-CF and CF mice were not detectable or subtle, and if they were more prominent as in the case of Cftr ^MHH mice, they were also existent in non-infected animals.

Forty-eight hours after challenge the numbers of viable P. aeruginosa TBCF10839 in the murine lungs ranged from zero to a maximum of 8.2 × 10³ CFU (Figure 6). The mice had cleared 99% or more of the initial inoculum. No differences could be observed between young and old and between CF and non-CF mice.

Examination of lung histology (Figure 7) revealed most signs of inflammation in the lung parenchyma such as alveolar histiocytosis, PMNs in the alveoli and partially even necrosis of the alveolar septae. In the most strongly affected animals approximately 20-30% of the tissue showed signs of an acute catarrhalic-suppurating alveolar pneumonia. Inflammatory cell infiltrates of bronchi and perivascular edema were seen in less than 10% of examined sections.

Among the different animal groups the young Cftr ^MHH mice exhibited a significantly higher inflammation than the old Cftr ^MHH mice in the lung parenchyma, but not in other lung compartments. Correspondingly young Cftr ^MHH mice had the highest mean histology score (Figure 8). At the chosen endpoint of 48 hours the number of viable bacteria in the lungs did not significantly correlate with the severity of airway inflammation classified by the histology score (Figure 6).