Age-associated inflammation and toll-like receptor dysfunction prime the lungs for pneumococcal pneumonia

Abstract

Background: Aging is associated with increased inflammation and risk of community-acquired pneumonia. Streptococcus pneumoniae co-opts the nuclear factor kappa B (NFkB)-regulated proteins polymeric immunoglobulin receptor (pIgR) and platelet-activating factor receptor (PAFr) to attach and invade cells. We sought to determine whether aging and chronic inflammation were associated with increased pIgR and PAFr levels in the lungs and increased susceptibility to S. pneumoniae infection.

Methods: Lung protein and messenger RNA levels were quantitated using Western blot and quantitative polymerase chain reaction. NFkB activation was measured by electrophoretic mobility shift assay. Cytokine levels were measured by cytometric bead analysis. To model chronic inflammation, mice were implanted with osmotic pumps that delivered tumor necrosis factor-alpha.

Results: Aged mice and those infused with tumor necrosis factor-alpha had increased levels of pIgR and PAFr in their lungs and were more susceptible to S. pneumoniae infection. During pneumonia, aged mice had reduced levels of pIgR and PAFr and less NFkB activation, despite greater bacterial burden. We determined that aged mice had decreased amounts of lung Toll-like receptors 1, 2, and 4 and reduced capacity to respond to S. pneumoniae with proinflammatory cytokine production.

Conclusions: Aged mice and, potentially, elderly humans are more susceptible to pneumonia because of a priming effect of chronic inflammation and Toll-like receptor dysfunction.

Figure 1. Aged mice are highly susceptible to S. pneumoniae infection

A) Bacterial titers in the lungs and blood of young and aged Balb/cBy mice one day after intranasal challenge with 10⁷ cfu of S. pneumoniae. B) Scoring of lung pathology in the same mice where 1 is no pathology on histological cross-section and 5 is extensive vascular congestion, hemorrhage and alveolar edema. C) Kaplan-Meier plot illustrating 100% mortality in aged mice at 36 hours compared to a 50% survival of young mice post-challenge. For A) and B) squares represent values obtained from individual mice. Horizontal bars indicate the median value. Statistical analysis was performed using a Student’s t-test. For C) statistical analysis was performed using a Kaplan-Meier Log-Rank Test.

Figure 2. Elevated levels of pIgR and PAFr in the lungs of aged mice

Relative amounts of A) pIgR and B) PAFr in the lungs of individual young (open square) and aged (closed square) mice as determined by quantitative chemiluminescent analysis of Western blots. C) Protein loads were determined to be equal based on subsequent blots for actin. Horizontal bars indicate the median value. Statistical analysis was performed using a Student’s t-test.

Figure 3. Young mice infused with TNFα express more pIgR and PAFr and are more susceptible to pneumonia

A) Silver stain demonstrating continuous delivery of intact TNFα by an osmotic pump in vitro. Relative levels of B) pIgR and C) PAFr in the lungs of mice receiving osmotic pump delivery of TNFα (closed diamond) or a saline loading control (open diamond) after five days; as determined by quantitative chemiluminescent analysis of Western blots. D) Protein loads were determined to be equal based on subsequent blots for actin. E) Bacterial titers in the lungs and blood of young Balb/cBy mice implanted with osmotic pumps that delivered TNFα or PBS two days after intranasal challenge with 10⁷ cfu of S. pneumoniae. Horizontal bars indicate the median value. Statistical analysis was performed using a Student’s t-test.

Figure 4. Levels of activated NFkB, pIgR and PAFr are lower in aged mice during an infection when compared to young mice

A) Relative levels of pIgR, B) PAFr, C) actin, and D) activated NFkB in young versus aged mice 2 days following infection with 10⁷ cfu of S. pneumoniae. Protein levels were determined by quantitative immunoblot analyses, activated NFkB levels determined by EMSA. Squares and diamonds indicate the individual protein expression level for each mouse tested. Horizontal bars indicate the median value. Statistical analysis was performed using a Student’s t-test.

Figure 5. TLRs are decreased only in aged mice prior to and during infection

Relative levels of TLR-1, TLR-2, and TLR-4 proteins measured in the lungs of healthy and infected young and aged mice two days after infection. Squares indicate the individual protein expression level for each mouse tested. Horizontal bars indicate the median value. Protein loads were confirmed to be equal by probing for actin (not shown). Statistical analysis was performed using a Student’s t-test.

Figure 6. Aged mice produce less TNFα and IL-6 when challenged with S. pneumoniae components

Levels of TNFα and IL-6 in BALF of young and aged mice 6 hours after intratracheal challenge with 100 µl of PBS containing either 10⁷ cfu equivalents of whole heat killed S. pneumoniae (young n=4, aged n=6), purified pneumococcal cell wall (10⁸ cfu equivalents; young n=7, aged n=7) or recombinant pneumolysin (1µg/mL; young n=5, aged n=5). Statistics was performed using a Student’s t-test.

Figure 7. Illustration explaining how age-associated inflammation and Toll-like receptor dysfunction increases the susceptibility of the elderly to pneumococcal pneumonia

Underlying disease and chronic age-associated inflammation cause expression of the host proteins pIgR and PAFr in the lungs which the pneumococcus uses to attach to and invade lung cells. Once the infection is established, TLR dysfunction results in a muted immune response to the bacteria and development of fulminate pneumonia with a high mortality rate.