Attenuated response of aged mice to respiratory Francisella novicida is characterized by reduced cell death and absence of subsequent hypercytokinemia

Abstract

Background: Pneumonia and pulmonary infections are major causes of mortality among the growing elderly population. Age associated attenuations of various immune parameters, involved with both innate and adaptive responses are collectively known as immune senescence. These changes are likely to be involved with differences in host susceptibility to disease between young and aged individuals.

Methodology/principal findings: The objective of this study was to assess potential age related differences in the pulmonary host response in mice to the Gram-negative respiratory pathogen, Francisella novicida. We intranasally infected mice with F. novicida and compared various immune and pathological parameters of the pulmonary host response in both young and aged mice.

Conclusions/significance: We observed that 20% of aged mice were able to survive an intranasal challenge with F. novicida while all of their younger cohorts died consistently within 4 to 6 days post infection. Further experiments revealed that all of the aged mice tested were initially able to control bacterial replication in the lungs as well as at distal sites of replication compared with young mice. In addition, the small cohort of aged survivors did not progress to a severe sepsis syndrome with hypercytokinemia, as did all of the young adult mice. Finally, a lack of widespread cell death in potential aged survivors coupled with a difference in cell types recruited to sites of infection within the lung confirmed an altered host response to Francisella in aged mice.

Figure 1. A small proportion of aged mice are able to survive an intranasal challenge with F. novicida.

Aged mice (22–24 months; n = 18) and young mice (8–12 weeks; n = 18) were infected intranasally with a dose of 397 CFU/20 µl to 500 CFU/20 µl of F. novicida and monitored for survival. Some of the aged mice survived the challenge (4/18) while all of the young mice succumbed to the infection. Data was plotted using a Kaplain-Meier curve and statistical analysis was determined by a log rank test (p = .0002).

Figure 2. Aged mice display an altered pulmonary pathology in response to F. novicda.

Young and aged mice were intransally infected with F. novicida. Lungs were harvested at various time points and H&E staining was performed to help assess the pathology associated with the course of infection. Panels A and D show mock lungs from young and aged mice, respectively. The arrow indicates perivascular mononuclear cells present in mock aged lung. Panels B and E depict the lungs of young and aged mice at 3 DPI. The asterisk is highlighting large foci of necrosis that is typical by 3 DPI in the lungs of young mice. These foci were notably absent in aged mice. However, small perivascular and peribronchial aggregates of viable mononuclear cells (arrow) were commonly found in the aged lungs. Panels C and F show young and aged lungs at 4 DPI while panel G exemplifies a lung from an aged mouse at 8 DPI. Both C and F are characterized by large foci of necrosis and lung consolidation (asterisk). At 8 DPI (G) in the aged mice there are several perivascular and peribronchial mononuclear aggregates and a few are also present at 4 DPI in moribund aged mice (F, arrow). Magnification 100X.

Figure 3. Bacterial burdens in aged mice are reduced early in the infection.

Young and aged mice were intranasally infected and their lungs, spleen and blood were harvested, processed, and plated in order to determine the bacterial burden (n = 3–4 mice per group). Panel A shows a significant reduction in the number of bacteria recovered from the lungs of aged mice as compared to young mice throughout the course of infection, especially at 1 and 3 DPI. Panel B indicates a reduction in systemic dissemination in the blood between young mice and aged mice that could be considered potentially moribund and aged mice that seemed to be able to control the infection. Panel C shows the bacterial burden in the spleen and similarly indicates a reduction in the amount bacteria recovered from aged mice (survivors and non-survivors) relative to young mice. * p<.05; **p<.005; ***p<.001.

Figure 4. Mediators associated with neutrophil chemotaxis and function are sharply reduced in aged mice.

Lungs were aseptically removed from young and aged mice (n = 3 to 4 mice per group) and processed to yield whole lung homogenates as described in the material and methods section. The homogenates were then analyzed using the Rodent MAP 2.0 from Rules Based Medicine (Austin, TX). CXCL6 (A), GM-CSF (B), and CXCL2 (C) are all cytokines important for neutrophil chemotaxis and were all less abundant in the lungs of both aged moribund mice and aged survivors. Myeoloperoxidase (D) was also determined to be reduced in the aged, especially in survivors at 3 DPI. *p<.05, **p<.005; ***p<.001.

Figure 5. Inflammatory cytokines associated with severe sepsis and hypercytokinemia are at lower levels in aged survivors.

Levels of inflammatory cytokines associated with severe sepsis were measured in the lungs of young and mice intranasally infected with F. novicida (n = 3 or 4 mice per group). Levels of IFN-gamma, IL-6, IL-1 alpha, TNF-alpha, CCL2 and CXCL10 were all elevated at 3 DPI in the young and remained significantly higher than their aged counterparts through 5 DPI. Strikingly, aged survivors had significantly reduced levels in most of these cytokines. * p<.05; **p<.005; p<.001.

Figure 6. Anti-inflammatory cytokines are also aberrantly upregulated in young mice but not in aged survivors.

The levels of anti-inflammatory cytokines were determined in the lungs of young and aged mice infected with F. novicida at 1, 3, and 5 days post infection (n = 3 or 4 mice per group). IL-10, IL-4, IL-11 and IL-5 (Panels A, B, C and D, respectively) were all determined to be upregulated to various degrees in young mice by 3 DPI. Aged survivors presented with significantly lower levels of IL-10, IL-4, and IL-11 at 3 DPI. * p<.05, ** p<.005, *** p<.001.

Figure 7. Extent of cell death correlates with outcome of respiratory tularemia in young and aged mice.

Young and aged mice were intranasally infected with F. novicida and their lungs were subsequently harvested and processed for in situ fluorescent TUNEL staining. In situ TUNEL staining was performed according to manufacturer's protocol with slight modifications. Panels A and D show TUNEL staining from mock lungs in young (Y) and aged (A) mice. Panels B and E are representative micrographs of young and aged mice at 3 DPI. Note the large necrotic infiltrate present at 3 DPI in the lungs of young mice (asterisk). Panels C and F are illustrating young and aged mice at 4 DPI, respectively. Lung consolidation and necrosis was present throughout the tissue in young mice as indicated by TUNEL (asterisk). Aged mice that appeared moribund at 4 DPI also showed a sharp increase in the extent of TUNEL positive areas (asterisk in F). Panel H represents a lung from an aged survivor at 8 DPI. The arrow indicates a perivascular aggregate. Strikingly this area, as well as the entire lung contains less TUNEL positive cells than moribund young and aged mice. Panel G is the quantification of TUNEL staining in a representative of 3 independent experiments. A significant increase in TUNEL was observed at 3 DPI and 4 DPI in the young mice as well as at 1 DPI and at 4 DPI in aged mice (n = 3 per group except 8 DPI where n = 2).