Genetically diverse aging mice are more susceptible to Mycobacterium tuberculosis than young
Mouse models of genetic diversity are becoming more widely used [4],[13],[22]-[25]. A majority of M. tuberculosis research in young and old mice has used the C57BL/6 inbred strain, with fewer studies using B6 hybrids or other inbred strains such as Balb/c, C3H/HeJ, DBA/2, CBA/J, I/St, and A/Sn [26]. Through these studies we have learned valuable information regarding responses to M. tuberculosis, characteristics of susceptible and resistant strains, and genes that contribute to susceptibility. However, the allelic homozygosity of inbred strains leads to expression of deleterious recessive genes, which may affect results especially in aging studies (1). Therefore, genetically diverse mouse models may be useful for M. tuberculosis research.
As with human beings, aging genetically diverse HET3 mice are more susceptible to aerosolized M. tuberculosis than young, with median survivals of 118 and 197 days in aging and young mice, respectively (Figure 1A). A characteristic of TB in mice and human beings is weight loss; in fact, weight is a useful indicator of TB in mice because it occurs before respiratory, motor, and social disturbances [27]. And because weighing mice is non-invasive, repeated measures can be performed on individual mice with little stress. In aging HET3 mice, weight loss began earlier in M. tuberculosis infection than in young mice (Figure 1B,C), even though aging mice were heavier (Figure 1D,E). Aging mice also lost relatively more weight due to TB (Figure 1F).
Evidence of clinical disease began on average, 21 days after M. tuberculosis infection in old mice; in contrast disease in young mice began, on average, 127 days after infection. This likely reflects better control of M. tuberculosis bacilli, or better control of detrimental inflammation, in young mice. The duration of disease was the same in aging and young HET3 mice (85 ± 50 versus 88 ± 41 days, respectively). Regardless of age, lungs eventually fill with inflammatory cells and variable necrosis in all mice (not shown). Therefore, the main effect of old age appears to be that disease onset occurs earlier.
We expected that body weight would positively correlate with survival. However, this was not true for aging HET3 mice (Figure 2A), but we did observe that the rate of weight loss was the best indicator of TB disease progression, shown and modeled by an exponential decay (Figure 2B). Although weight changes do not reflect specific immunologic or pathologic changes, the ability to track body weight provides a foundation to identify biomarkers that precede weight loss.
We explored relationships between survival and immunity that may be important for resistance to M. tuberculosis. Survival did not correlate with the total (not shown) or naïve CD4 T cell numbers prior to infection (Figure 2C). Survival correlated significantly with ESAT-6 specific IFN-γ in HET3 mice but the correlation was weak (Figure 2D) and some mice were clear exceptions.
In summary, our findings suggest that aged, genetically diverse mice can model age-related susceptibility to M. tuberculosis primary infection in humans. Although T cell responses are important for control of M. tuberculosis (shown by markedly increased susceptibility when these molecules are absent in inbred mice [28],[29]), in immune competent aged HET3 mice, antigen-specific IFN-γ is a positive but weak correlate of survival.
Rapamycin boosts antigen-specific interferon-gamma responses
Interventions to improve the length and quality of life are of interest, and HET3 mice are advantageous for research and testing interventions because the population of siblings is reproducible and results can be compared across time and from different laboratories [13],[19],[30]. Furthermore, rapamycin benefits multiple types of immune responses in old mice but not young mice (unpublished, DH), and rapamycin could improve outcome to M. tuberculosis infection by enhancing innate or acquired immunity. Rapamycin has beneficial effects on lifespan [11]-[13] and controls metabolism of immune cells [14] important for resistance to M. tuberculosis[28],[29]. Rapamycin also stimulates M. tuberculosis antigen-specific TH1 cells [15],[31] by inducing autophagy [32] which can eliminate intracellular M. tuberculosis bacilli [33]. We thus tested whether rapamycin enhanced immune responses to M. tuberculosis in aging mice. Indeed, rapamycin increased ESAT-6-specific IFN-γ responses during M. tuberculosis infection (Figure 3A). Rapamycin also increased ESAT-6-specific proliferation, the numbers of IFN-γ producing cells, and amount of IFN-γ produced by the responding cells (Figure 3B-D) when ESAT-6 was administered as a vaccine. Whether these responses to ESAT-6 vaccination improve control of M. tuberculosis bacillary growth or prolong survival remains to be determined.
In summary, our results indicate that oral rapamycin showed trends for enhancing ESAT-6 specific responses in genetically diverse old mice. The variable statistical significance likely reflects the relatively small sample size, in particular with the vaccination studies in DO mice. Although additional studies are needed, the finding may be relevant for elderly people because oral delivery of agents is an attractive method to improve immune function.
Rapamycin does not improve survival of aging mice with primary M.tb infection
We next assessed whether rapamycin treatment improved survival, delayed the onset of TB disease, or altered the lung lesions during primary M. tuberculosis infection. Rapamycin did not prolong survival or extend the median survival (Figure 4A) or delay disease onset (not shown). Modest changes in lungs are attributable to rapamycin: a slight reduction in the proportion of mice with marked necrosis and neutrophil influx and a shift toward increased lymphocytes (Figure 4B). Regardless, lung damage in all mice was substantial, and thus, despite having potential benefits (on lifespan, cellular and humoral immunity, autophagy), rapamycin in our model did not delay TB disease or extend survival in M. tuberculosis infected aged HET3 mice regardless of immunologic changes or tissue architectural changes.
We observed a trend for rapamycin-enhanced antigen-specific responses (proliferation, IFN-γ) in the vaccination model. It is unknown whether rapamycin or the enhanced immune responses can actually protect against M. tuberculosis challenge in this model, but this is a logical next step for future studies. However, clinical outcomes and survival measures in mice are now even more important when testing interventional or preventative therapies against M. tuberculosis infection because protective correlates of immunity are not fully known [16]-[18]. This strategy is necessary to assess the potential of rapamycin to improve vaccination efficacy in elderly people.