Aging exacerbates mortality of Acinetobacter baumannii pneumonia and reduces the efficacies of antibiotics and vaccine

Enhanced mortality in aged mice after respiratory A. baumannii infection

To determine whether susceptibility to A. baumannii infection was increased with advanced age, young and aged mice were infected by non-invasive intratracheal inoculation of LAC-4 (5×10⁶ CFU). Survival rate and clinical score of mice were monitored for 7 days. After infection, the clinical signs of aged mice were more severe than those of young mice (Figure 1A). All aged mice appeared very sick, moved slowly, and hunched at 24 hours post infection (hpi). Mortality of aged mice was 100% within 48 hours after LAC-4 infection, whereas 60% of young mice survived (Figure 1B). The results indicate aged mice are more susceptible to respiratory A. baumannii infection than young mice.

Figure 1. Increased susceptibility to A. baumannii infection in aged mice. Young (n = 10) and aged mice (n = 10) were infected intratracheally with 5×10⁶ CFU of LAC-4. (A) Clinical score and (B) survival rate of mice were monitored for 7 days. Clinical score is expressed as means ± SEM. The data represent one of 2 independent experiments. Survival curves were compared using log-rank test. Statistical significance of clinical score was determined by Student’s t test. **, P < 0.01.

Increased bacterial burdens and lung injury in aged mice

Next, bacterial burdens in the lung, blood, and spleen at 24 hpi were evaluated. Aged mice had more than 10-fold higher bacterial burdens in lungs compared with young mice. More importantly, aged mice had remarkably increased bacterial burdens in blood and spleen compared with young mice, showing higher level of extrapulmonary dissemination of bacteria after infection (Figure 2A).

Figure 2. Increased bacterial burdens and lung damage after A. baumannii infection in aged mice. Young and aged mice were infected intratracheally with 5×10⁶ CFU of LAC-4 and the lung, blood, and spleen were collected at 0 h and 24 hpi. (A) Bacterial burdens in the lung, blood, and spleen of young and aged mice at 24 hpi were counted on TSA plates. (B) Lung histopathology of young and aged mice were observed at 0 h and 24 hpi (magnification=200×). (C) Serum albumin levels in BALF of young and aged mice were determined by ELISA. Data are presented as mean ± SEM of five mice per condition and represent one of 2 independent experiments. Statistical analyses were performed by Student’s t test. *, P < 0.05, **, P < 0.01.

We next evaluated lung injury after LAC-4 infection by histological analysis. As shown in Figure. 2B, the lung histology of aged mice was comparable to that of young mice before infection (0 h). Whereas, histopathological changes of lungs in aged mice were more severe than those in young mice at 24 hpi. Lungs of young mice showed limited tissue destruction with mild infiltration of perivascular and interstitial inflammatory cells. In contrast, lungs of aged mice appeared more severe and extended lesions with extensive accumulation of inflammatory cells (Figure 2B). Serum albumin in bronchoalveolar lavage fluid (BALF) is an indicator for assessing the epithelial barrier permeability of lung. Aged mice exhibited higher levels of serum albumin in BALF at 24 hpi than young mice (Figure 2C), indicating that the epithelial barrier of aged mice was destroyed more severely than that of young mice after infection. These results suggest A. baumannii infection causes more severe lung damage in aged mice.

Elevated inflammatory cytokine responses in aged mice after A. baumannii infection

Next, we evaluated inflammatory cytokine levels in response to A. baumannii infection in aged and young mice. The basal levels of TNF-α, IL-1β, and IL-6 in BALF and serum were comparable between aged and young mice. At 24 hpi, TNF-α and IL-6 levels in serum and BALF from aged mice were significantly higher than those from young mice. However, there was no significant difference of IL-1β levels in BALF and serum between infected young and aged mice (Figure 3A and B). In addition, TNF-α and IL-6 mRNA expression in the lungs from infected aged mice were significantly higher than those from infected young mice, whereas mRNA expression of IL-1β exhibited similar fold change in infected young and aged groups (Figure 3C). The data suggest aging results in a markedly local and systemic inflammation characterized by enhanced TNF-α and IL-6 expression.

Figure 3. Enhanced levels of cytokines after A. baumannii infection in aged mice. Young and aged mice were infected intratracheally with 5×10⁶ CFU of LAC-4. At 0 h and 24 hpi, serum and BALF were collected and mRNA was isolated from lungs. TNFα, IL-6, and IL-1β levels in serum (A) and BALF (B) were detected by ELISA. (C) TNFα, IL-6, and IL-1β mRNA expression in lungs were detected by real-time PCR. Data are presented as mean ± SEM of five mice per condition and represent one of 2 independent experiments. Statistical analyses were performed by Student’s t test. *, P < 0.05, **, P < 0.01.

Increased inflammatory cell infiltration in BALF in aged mice after infection

In BALF, there was no obvious difference in the total cell numbers between uninfected young and aged mice (0 h). The main cells were macrophages before infection (Figure 4C), whereas the neutrophils and monocytes were barely detected (Figure 4B and D). At 24 hpi, the total cell numbers increased in both young and aged mice compared to uninfected mice and aged mice had significantly higher total cell numbers than young mice (Figure 4A). At 24 hpi, neutrophils were the dominant cells in BALF and the numbers of neutrophils in aged mice were approximately 1.5-fold higher than those in young mice (Figure 4D). Taken together, aged mice have enhanced inflammatory cell infiltration after respiratory A. baumannii infection.

Figure 4. Elevated numbers of inflammatory cells after A.baumannii infection in aged mice. Young and aged mice were infected intratracheally with 5×10⁶ CFU LAC-4 and BALF were collected at 0 h and 24 hpi. Number of total cells (A), monocytes (B), macrophages (C), and neutrophils (D) in BALF of young and aged mice were determined. Data are presented as mean ± SEM of five mice per condition and represent one of 2 independent experiments. Statistical analyses were performed by Student’s t test. **, P < 0.01.

Enhanced chemokine production in aged mice in response to A. baumannii infection

To determine whether increased inflammatory cell infiltration in aged mice is associated with increased chemokine expression, mRNA levels of chemokine in lungs were detected by real-time PCR. The results showed there is no difference of chemokine mRNA expression between uninfected young and aged mice. At 24 hpi, higher mRNA expression of CXCL1, CCL2, and CCL7 in lungs were detected in aged mice than those in young mice (Figure 5). There was no obvious difference in mRNA levels of CXCL2 and CXCL5 between infected young and aged mice. The results suggest increased inflammatory cells are associated with increased levels of CXCL1, CCL2, and CCL7 expression.

Figure 5. Increased chemokines mRNA expression after A.baumannii infection in aged mice. Young and aged mice were infected intratracheally with 5×10⁶ CFU of LAC-4. At 0 h and 24 hpi, mRNA in lungs was isolated. CXCL1, CCL2, CCL7, CXCL2, and CXCL5 were measured by real-time PCR. Data are presented as mean ± SEM of four or five mice per condition and represent one of 2 independent experiments. Statistical analyses were performed by Student’s t test. *, P < 0.05, **, P < 0.01.

Decreased bactericidal ability of inflammatory cells in aged mice

Usually, inflammatory cells are regarded as the first line of host defense against infection. Our data showed aged mice have stronger inflammatory response to A. baumannii infection. But aged mice still had higher bacterial burdens than young mice. Thus, we assumed that the bactericidal abilities of inflammatory cells in aged mice might be impaired. We detected myeloperoxidase (MPO) levels in lungs and reactive oxygen species (ROS) activity of the neutrophils. The results showed basal MPO levels in lung homogenates are similar between uninfected young and aged mice. But MPO levels at 24 hpi were significantly lower in aged mice than in young mice (Figure 6A). In addition, ROS-positive neutrophils in BLAF of aged mice were significantly lower than that in young mice after infection (Figure 6B). These results indicate the bactericidal activity of inflammatory cells is reduced with aging, which might be responsible for the enhanced susceptibility to A. baumannii infection.

Figure 6. Bactericidal ability of inflammatory cells in young and aged mice. Young and aged mice were infected intratracheally with 5 × 10⁶ CFU of LAC-4. (A) MPO levels in the lung homogenate supernatants at 0 h and 24 hpi were detected by ELISA. (B) ROS production in neutrophils (CD11b⁺ Ly6G⁺ cells) in BALF at 24 hpi was stained by a ROS detection reagent (carboxy-H₂DCFDA) and assessed by flow cytometry. Data are presented as mean ± SEM of four or five mice per condition and represent one of 2 independent experiments. Statistical analyses were performed by Student’s t test. *, P<0.05, **, P<0.01.

Decreased efficacy of antibiotic therapy against A. baumannii in aged mice model

Next, we tried to evaluate whether aging affects the efficacy of antibiotics against A. baumannii infection. Young and aged mice were intratracheally inoculated with lethal dose of LAC-4 (1.5×10⁷ CFU). At 3 hpi, mice were treated with imipenem/cilastatin or saline twice a day. All young and age mice treated with saline developed severe clinical signs and died 100% within 48 hours. Whereas, young mice treated with antibiotics developed mild clinical signs and 100% survived the infection. In contrast, aged mice after antibiotic treatment showed more severe clinical signs than treated young mice (Figure 7A). Only 40% of aged mice treated with antibiotics survived (Figure 7B). In conclusion, these data suggest aging reduces the efficacy of antibiotics against A. baumannii.

Figure 7. Efficacy of imipenem/cilstatin treatment on respiratory A.baumannii infection. Young and aged mice were challenged intratracheally with 1.5×10⁷ CFU of LAC-4. At 3 hpi, groups of five mice were i.p. injected with imipenem/cilstatin (20mg/20mg)/kg/day or saline twice a day. (A) Clinical score and (B) survival rate of mice were monitored for 7 days. Clinical score is expressed as means ± SEM. The data represent one of 2 independent experiments. Survival curves were compared using log-rank test. Statistical significance of clinical score was determined by Student’s t test. *, P < 0.05, **, P < 0.01 antibiotic-treated aged mice versus antibiotic-treated young mice.

Reduced protection of vaccination against A. baumannii infection in aged mice

Next, we evaluated whether aging affects the vaccine efficacy against A. baumannii infection. Both young and aged mice were immunized with formalin-fixed LAC-4 (ffLAC-4) intramuscularly and then challenged with lethal dose of LAC-4 intratracheally. Unvaccinated young and aged mice died within 48 hpi. Vaccinated young mice showed milder clinical signs compared with unvaccinated young mice (Figure 8A). The survival rate of vaccinated young mice was 100% (Figure 8B), indicating that ffLAC-4 immunization induces protective response against A. baumannii infection in young mice. Whereas vaccinated aged mice were very sick and moved slowly after infection, showing the similar clinical scores to unvaccinated aged mice (Figure 8A). Vaccinated aged mice all died within 48 hpi (Figure 8B). These results indicate aging significantly reduces the protection induced by vaccination.

Figure 8. Protection of ffLAC-4 vaccination against respiratory A. baumannii infection. Groups of five young and aged mice were immunized intramuscularly with 1×10⁷ CFU of ffLAC-4 or PBS on day 0. On day 7, mice were challenged intratracheally with 1.5×10⁷ CFU of LAC-4. (A) Clinical score and (B) survival rate of mice were monitored for 7 days after infection. Clinical score is expressed as means ± SEM. The data represent one of 2 independent experiments. Survival curves were compared using log-rank test. Statistical significance of clinical score was determined by Student’s t test. **, P < 0.01 immunized aged mice versus immunized young mice.

Mice

Young (6-8 weeks) and aged (18-21 months) female C57BL/6 mice were purchased from the Animal Center of Chongqing Medical University. The mice were kept under pathogen-free conditions. All procedures were approved by the Animal Ethical and Experimental Committee of the Amy Medical University.

Pneumonia model and samples collection

A clinical A. baumannii strain LAC-4 was kindly provided by Professor Chen [37]. The bacteria were grown in tryptone soy broth at 37 °C. At mid-log-phase, bacteria were collected and suspended in phosphate buffer saline (PBS). Mice were anaesthetized by intraperitoneal injection of pentobarbital sodium (62.5 mg/kg of body weight) and then infected with LAC-4 in 20 μl PBS by non-invasive intratracheal inoculation under direct vision adapted as previously described [38]. The infection dose was confirmed by plating 10-fold serial dilutions on tryptone soy agar (TSA) and counting bacteria colony-forming unit (CFU). The survival rate and clinical score of mice were monitored daily for 7 days (n = 10). The clinical scores were recorded as described previously [37]: 0, normal clinical signs; -1, slightly ruffled fur but active; -2, ruffled fur, slow movement, and sick; -3, ruffled fur, hunched posture, squeezed eyes, and very sick; -4, moribund; -5, dead. At designated time, groups of five mice were sacrificed and bacterial burdens in the lung, blood, and spleen were determined. Serum was separated from blood samples and stored at -80 °C. Excised lungs were used for histopathology. Collection of BALF was performed by flushing of the lungs with 0.5 ml cold PBS for 3 times. The supernatant of BALF was obtained and stored at -80 °C. BALF cells were analyzed by flow cytometry.

Quantitative bacteriology and histopathology

Lungs and spleens were homogenized in sterile PBS. Bacteria from organ homogenates or blood were determined by 10-fold serial dilutions and were cultured on TSA plates. CFUs were counted 18 h thereafter and results were expressed as log₁₀ CFU per organ or per milliliter blood. For histopathology, excised lungs were fixed immediately in 4% formalin and embedded in paraffin. Sections were cut in 5 μm thick, stained with hematoxylin-eosin and observed by a light microscope.

Cytokine and protein analysis

TNF-α, IL-1β, and IL-6 concentrations in serum and BALF were detected using ELISA kits (mouse TNF-α ELISA max standard set, mouse IL-1β ELISA max standard set, and mouse IL-6 ELISA max standard set, Biolegend). Serum albumin in BALF was assessed by mouse albumin ELISA quantitation set (Bethyl Laboratories). MPO levels in lung homogenates were quantified by mouse myeloperoxidase duoset ELISA Kit (R&D Systems). ELISAs were performed following the manufacturer's instructions.

Quantitative real-time PCR

Total RNA of lungs was isolated by RNA iso Plus (Takara) and reverse transcribed with PrimeScript^™ RT reagent Kit (Takara). Gene expression was detected using SYBR green Premix (Takara) with specific primers on CFX96 real-time PCR detection machine (Bio-Rad). The primer sequences used were as follows: TNF-α forward primer: 5′-CCTATGTCTCAGCCTCTT CTCAT-3′, and reverse primer: 5′-CACTTGGTGGTTT GCTACGA-3′; IL-1β forward primer: 5′-GGACCCC AAAAGATGAAGGGCTGC-3′, and reverse primer: 5′-GCTCTTGTTGATGTGCTGCTGCG-3′; IL-6 forward primer: 5′-CCTCTCTGCAAGAGACTTCC-3′, and reverse primer: 5′-CTCCGGACTTGTGAAGTAGG-3′; CXCL2 forward primer: 5′-AGGGCGGTCAAAAAG TTTGC-3′, and reverse primer: 5′-CAGGTACGATCC AGGCTTCC-3′; CXCL5 forward primer: 5′-TGGCA TTTCTGTTGCTGTTC-3′, and reverse primer: 5′-CACCTCCAAATTAGCGATCAA-3′; CCL2 forward primer: 5′-TTAAAAACCTGGATCGGAACCAA-3′, and reverse primer: 5′-GCATTAGCTTCAGATTTA CGGGT-3′; CCL7 forward primer: 5′-CCACCATG AGGATCTCTGC-3′, and reverse primer: 5′-TTGACA TAGCAGCATGTGGAT-3′; β-actin forward primer: 5′-GGCTGTATTCCCCTCCATCG-3′, and reverse primer: 5′-CCAGTTGGTAACAATGCCATGT-3′. The ΔΔCt method was used to calculate the relative gene expression with β-actin as the housekeep-gene. The results were expressed as relative fold changes compared to non-infected mice.

Flow cytometric analysis

Cell suspensions from BALF were blocked with rat serum for 15 min. Cells were then stained with 50 μl cocktail of fluorophore-conjugated antibodies in the dark at 4 °C for 30 min. The monoclonal antibodies were as follows: CD45-PE/Cy7 (30-F11), CD11b-PerCP/Cy5.5 (M1/70), CD11c-APC (N418), Ly6G-FITC (1A8) (Biolegend). Labeled cells were analyzed on a BD FACSCanto™ II flow cytometer (BD Biosciences). The cell populations in BALF were identified as described previously [39]: CD45⁺CD11b⁺CD11c⁻Ly6G⁺ cells were considered to be neutrophils; CD45⁺CD11b⁺CD11c⁻Ly6G^lowcells were considered to be monocytes; CD45⁺CD11b⁻CD11c⁺Ly6G⁻ cells were considered to be macrophages.

Intracellular ROS assay

Groups of four or five mice were infected intratracheally with A. baumannii and BALF cells were collected at 24 hpi. The isolated cells were resuspended in prewarmed hank's balanced salt solution (HBSS) containing 1 μM carboxy-H₂DCFDA (Invitrogen) for 30 min at 37 °C, 5% CO₂. For a negative control, cells were incubated with HBSS only. The reaction was stopped by moving the cells onto ice. Cells were stained for CD11b and Ly6G and analyzed by flow cytometry.

Antibiotic treatment

Groups of five young and aged mice were intratracheally infected with 1.5×10⁷ CFU of LAC-4 and treated with imipenem /cilastatin ((20mg/20mg)/kg of body weight/day, twice a day, i.p.) or saline starting 3 h after inoculation for 3 days. Survival rate and clinical score of mice were observed for daily 7 days.

Vaccination

The ffLAC-4 prepared as described was used as a vaccine [37]. Groups of five young and aged mice were intramuscularly immunized with 1×10⁷ CFU of ffLAC-4 in 250 μl PBS or PBS only at day 0. At day 7, the mice were intratracheally challenged with 1.5×10⁷ CFU of LAC-4 in 20 μl PBS. Survival rate and clinical score of mice were observed daily for 7 days.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism version 6.0. Survival curves were analyzed by log-rank tests. In all other studies, differences between young and aged groups were analyzed by Student’s t test. Values are expressed as mean ± SEM. A p value of less than 0.05 was considered statistically significant.