Sirolimus alters lung pathology and viral load following influenza A virus infection

Sirolimus was purchased from MedChem Express, LLC (Princeton, NJ). The drug was dissolved in dimethyl sulfoxide (DMSO) at 50 μg/μL (55 mM) and stored in at −20 °C. This concentrated stock was diluted to a final concentration of 0.5 μg/μL in dH₂O immediately before use. Ketamine (50 mg/mL), xylazine (suspended in methanol at 10 mg/mL), and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Ketamine-xylazine solution consisted of 80 μL (4 mg) ketamine plus 20 μL (0.2 mg) xylazine and stored at 4 °C for a maximum of 2 weeks. The solution was diluted 1:10 with dH₂O immediately prior to use. Mice were injected intraperitoneally with 10 μL/g (40 μg/g ketamine +2 μg/g xylazine) before inoculation and other surgical procedures. Adequate sedation was confirmed before intranasal inoculations by the absence of footpad reflexes. Mice were held in a supine position (at a 45° angle) with the back supported by the palm and the neck skin fold by the thumb and index finger. The inoculum was slowly released from a 10-μL micropipette as two small drops covering the two nostrils. The mice were allowed to inhale the volume without forming bubbles. They were then maintained in the same position until they regained consciousness and their rapid breathing returned to normal.

BALB/c mice (4–8 weeks old; Jackson Laboratory, Bar Harbor, ME) were housed at 22 °C and 60% humidity. They had ad libitum access to standard rodent chow and filtered water. The experiments received approval from the Animal Ethics Committee-UAE University-College of Medicine and Health Sciences (Protocol No. A3–13).

Mice were anesthetized by the ketamine-xylazine mixture and intranasally inoculated (as conferred above) with 10^8.1 TCID₅₀ per nostril of IAV A/PR/8/34 (H1N1) on day 0 [8]. The mice were also injected intraperitoneally with either sirolimus [14] or an equal volume of the vehicle DMSO, both administered for 5 uninterrupted days every week for a total of 4 weeks beginning on the day of inoculation (day 0). Sirolimus solution was prepared immediately before use by diluting the 50 μg/μL original stock (in DMSO) with dH₂O to a final concentration of 0.5 μg/μL. The sirolimus dose was 5 μg/g (10 μL/g). DMSO was also diluted 100-fold with dH₂O and injected at 10 μL/g (actual DMSO dose, 0.1 μL/g).

Specimens were collected as previously described [8]. For viral RNA extraction, one lung specimen was homogenized in TRIZOL (Invitrogen, Grand Island, NY) and the supernatant was stored at −80 °C. For histology, lung specimens were fixed in 4% phosphate-buffered paraformaldehyde and embedded in paraffin. Sections of the fixed tissue (5–7 μm thickness) were stained with hematoxylin and eosin (H&E) and examined under a light microscope.

A scoring system previously validated for respiratory syncytial virus infection was used to grade the intensity of disease [15, 16]. Histology sections were graded as follows: (A) degree of peribronchiolar/bronchial infiltrate (none, <25%, 25–75%, >75%); (B) type of peribronchiolar/bronchial infiltrate (none, interrupted collar, complete collar <5 cells thick, complete collar >5 cells thick); (C) bronchiolar/bronchial luminal exudate (none, ≤25% luminal occlusion, ≥25% luminal occlusion); (D) perivascular infiltrate (none, <10%, 10–50%, >50%); and (E) parenchymal pneumonia (none, patchy parenchymal infiltrate, heavy parenchymal infiltrate). The total score (ranged from 0 to 26) was set as: [A + 3(B + C) + D + E], Table 1.

Viral RNA was detected in the lung tissue of infected mice via RT-PCR [17]. Lungs were homogenized in 1.0 mL TRIZOL (Invitrogen, Carlsbad, CA), and cellular RNA was isolated from the supernatant by chloroform and isopropyl alcohol extraction. cDNA was prepared from two μg RNA using M-MLV Reverse Transcriptase (Promega, Madison, Wis.). Real-time PCR was performed to detect IAV matrix gene using previously described primers and probes [17], TaqMan® Universal PCR Master Mix and Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System (Foster City, CA). Amplification conditions were: an initial denaturation step at 94 °C for 5 min followed by 40 cycles of denaturation at 94 °C for 45 s and annealing at 60 °C for 1 min. Standard curve was prepared using cDNA from the virus sample (10^8.1 TCID₅₀).

Invasive pulmonary function analysis was performed using the FlexiVent instrument from SCIREQ (Montreal, PQ, Canada), as previously described [18]. This forced oscillation system measured respiratory disease in tracheotomized mice via changes in thoracic resistance (Rrs, cmH₂O.s/mL), thoracic compliance (Crs, mL/cmH₂O), large airway resistance (Rn, cmH₂O.s/mL), lung tissue damping (resistance, G, cmH₂O/mL), and lung tissue elastance (H, cmH₂O/mL) at baseline and after methacholine challenges. Methacholine responsiveness was shown as area under the curve (AUC) of Rrs, Crs and Rn against methacholine concentration (0, 0.6, 1.25, 2.5, 5, and 10 mg/mL). Airway obstruction was also evaluated using the fast-flow maneuvers forced expiratory volume at 50 ms (FEV0.05, in mL).

The impact of sirolimus administration on the severity of IAV-induced body weight changes was monitored over 25 days. Mice were intranasally inoculated with IAV on day 0. Groups of IAV-infected mice were injected intraperitoneally with either sirolimus or an equal volume of the vehicle DMSO, both administered for 5 uninterrupted days every week for a total of 4 weeks beginning on the day of inoculation (day 0). Uninfected naïve mice administered either sirolimus or DMSO served as controls. As expected, uninfected control mice treated with either DMSO or sirolimus slowly gained weight over time. IAV-induced weight loss peaked between days 6 and 11 post-infection; averaging 12% in IAV-infected mice that were administered sirolimus (p = 0.002 [compared to DMSO or sirolimus alone]), in comparison to 5% in mice that received DMSO (p = 0.017, [compared to DMSO] or p = 0.030 [compared to IAV + sirolimus]), Fig. 1. Thus, the body weight loss was more pronounced in IAV-infected mice that were administered sirolimus than that in mice received IAV alone.

In order to determine the impact of sirolimus administration on IAV replication, we monitored viral copy numbers at selected time points following IAV infection. Figure 2 shows the viral copies on days 4, 10 and 25 post-infection. Natural log viral gene copies on day 4 in IAV-infected mice that were administered sirolimus were similar to those in mice that received IAV alone (p = 0.132). However, the number of copies on day 10 in IAV-infected mice that were administered sirolimus were approximately 13-fold higher than in mice that received IAV alone (p = 0.008). Viral gene copies were not detected on day 25 in either group.

In order to determine the impact of sirolimus administration on the severity of pulmonary disease, we monitored lung histology following IAV infection. As expected, mice that received DMSO did not exhibit any peribronchial inflammation and pulmonary parenchymal architecture was preserved. In contrast, lung histology in mice received sirolimus alone showed focal interstitial thickening and inflammation on day 4, and mild peribronchial inflammation on day 10 (Fig. 3, arrows). On days 4, 10, and 25 post-infection, lung findings in mice infected with IAV showed various degrees of patchy peribronchial and perivascular inflammation with dense, diffuse parenchymal inflammation and formation of lymphoid nodules. IAV-infected mice treated with sirolimus also showed somewhat similar patchy peribronchiolar inflammation with increased peribronchial inflammation (patchy and interrupted, most prominent on day 10 post-infection), Fig. 3. Lung inflammation reached a disease score of 9.0 ± 4.5 in IAV-infected mice that were administered sirolimus, as compared to 11.5 ± 4.5 in mice that received IAV alone (p = 0.335), Table 1.

In order to determine the impact of sirolimus administration on pulmonary function, we monitored lung function changes following IAV infection at selected time points post-infection. Invasive pulmonary function analysis was performed using the FlexiVent instrument, as previously described [18]. This forced oscillation system assessed the respiratory disease in tracheotomized mice by measuring thoracic resistance (Rrs), thoracic compliance (Crs), large airway resistance (Rn), lung tissue damping (resistance, G), and lung tissue elastance (H). These parameters were determined at baseline and after methacholine challenge (Fig. 4). In addition, fast-flow maneuvers, such as forced expiratory volume at 50 ms (FEV0.05) were also determined (Table 2). Impaired lung function was evident in IAV-infected mice on days 4 and 10, mainly showing increased thoracic and large airway resistances and decreased thoracic compliance. The changes (at both baseline and after methacholine) were less pronounced on day 4 and more severe on day 10 in IAV-infected mice that were administered sirolimus (Fig. 4).