TLR-2 independent recognition of Mycobacterium tuberculosis by CD11c+ pulmonary cells from old mice.

The elderly are particularly susceptible to infectious diseases such as influenza, bacterial pneumonia, and tuberculosis. Current vaccines are only partially protective in old age, which makes the elderly a critical target group for the development of new vaccine strategies. The recognition of pathogens via toll like receptors (TLR) and the subsequent generation of pro-inflammatory cytokines has generated interest in incorporating TLR agonists into new vaccines to enhance immunogenicity. However, TLR function is reportedly decreased in old age, leading to questions regarding the benefit of including TLR agonists into vaccines for the elderly. It is critical that we understand the function and role of TLRs in aged hosts prior to approving new TLR based adjuvants for vaccines that will be delivered to the elderly. In this study we determine the ability of TLRs on pulmonary macrophages from old mice to recognize and respond to infection with the virulent pathogen Mycobacterium tuberculosis (M. tb). Although pulmonary (CD11c(+)) cells from old mice were fully capable of producing cytokines in response to M. tb infection, we demonstrate that in contrast to young mice, M. tb induced cytokine production occurred independently of TLR-2. Our data indicate that the inclusion of TLR-2 agonists into new vaccines may not be fully effective in the elderly population. Investigation into such age-related differences in TLR function is of critical importance for the design of effective vaccines that will protect the elderly against infectious diseases.

CCL5 participates in early protection against Mycobacterium tuberculosis.

Control of M.tb, the causative agent of TB, requires immune cell recruitment to form lung granulomas. The chemokines and chemokine receptors that promote cell migration for granuloma formation, however, are not defined completely. As immunity to M.tb manifests slowly in the lungs, a better understanding of specific roles for chemokines, in particular those that promote M.tb-protective T(H)1 responses, may identify targets that could accelerate granuloma formation. The chemokine CCL5 has been detected in patients with TB and implicated in control of M.tb infection. To define a role for CCL5 in vivo during M.tb infection, CCL5 KO mice were infected with a low dose of aerosolized M.tb. During early M.tb infection, CCL5 KO mice localized fewer APCs and chemokine receptor-positive T cells to the lungs and had microscopic evidence of altered cell trafficking to M.tb granulomas. Early acquired immunity and granuloma function were transiently impaired when CCL5 was absent, evident by delayed IFN-gamma responses and poor control of M.tb growth. Lung cells from M.tb-infected CCL5 KO mice eventually reached or exceeded the levels of WT mice, likely as a result of partial compensation by the CCL5-related ligand, CCL4, and not because of CCL3. Finally, our results suggest that most T cells use CCR5 but not CCR1 to interact with these ligands. Overall, these results contribute to a model of M.tb granuloma formation dependent on temporal regulation of chemokines rather than on redundant or promiscuous interactions.

Interleukin-12 is sufficient to promote antigen-independent interferon-gamma production by CD8 T cells in old mice.

Numerous functional defects have been identified in naive T cells from aged mice, including deficiencies in proliferation, cytokine production and signal transduction. It is well documented that the ratio of naïve to memory T cells significantly decreases with age resulting in the majority of T cells from aged hosts expressing activated/memory T-cell markers (CD44(hi)), yet it is unclear whether T cells with a CD44(hi) phenotype in aged hosts are functionally equivalent to T cells with a similar phenotype in young hosts. We have identified a population of CD44(hi) CD8 T cells in old mice that are capable of secreting interferon-gamma (IFN-gamma) in response to interleukin-12 (IL-12) stimulation. This occurred in the absence of T-cell receptor engagement, a function that was not observed in CD8 T cells from young mice. This phenotype was associated with increased IL-12 receptor beta2 gene expression and IL-12 induced signal transducer and activator of transcription 4 (STAT-4) activation, even when CD8 T-cell numbers from young and old mice were normalized for CD44(hi) expression. Furthermore, we demonstrate that IL-12-induced STAT-4 activation was required for T helper type 1 (Th1) cytokine-induced IFN-gamma production in CD8 T cells. These data illustrate that old mice possess a specialized subset of CD44(hi) CD8 T cells with an enhanced responsiveness to IL-12, enabling these cells to produce substantial amounts of IFN-gamma in response to Th1 cytokine stimulation. We have therefore identified a functional difference in the populations of CD44(hi) CD8 T cells from young and old mice, and believe that understanding age-associated immunological changes is essential for helping the elderly combat deadly diseases.

CD8 T Cells in old mice contribute to the innate immune response to Mycobacterium tuberculosis via interleukin-12p70-dependent and antigen-independent production of gamma interferon.

Elderly individuals have increased morbidity and mortality associated with infectious diseases due in part to the progressive age-associated decline in immune function. Despite this, the old mouse model of Mycobacterium tuberculosis infection has revealed a CD8- and gamma interferon (IFN-gamma)-dependent early resistance to infection. In this study, we investigated the mechanism by which CD8 T cells from old mice contributed to the early immune response to M. tuberculosis. Following a low-dose aerosol infection with M. tuberculosis, CD8 T cells were identified as being a dominant source of IFN-gamma expression in the lungs of old mice early after infection, before the typical onset of antigen-specific immunity. In addition, M. tuberculosis-induced IFN-gamma production by CD8 T cells isolated from naïve old mice was major histocompatibility complex class I independent but was dependent on interleukin-12p70, confirming an innate role of CD8 T cells during M. tuberculosis infection. Moreover, the ability of CD8 T cells from old mice to produce increased innate IFN-gamma levels in response to M. tuberculosis infection was defined as a unique function of CD8 T cells from old mice and not the aged lung environment. Finally, we have identified increased expression of SET as being one possible mechanism by which CD8 T cells from old mice produce enhanced levels of IFN-gamma. Additional characterizations of the signaling events that lead to enhanced innate IFN-gamma production by CD8 T cells in old mice may lead to novel strategies to further enhance or perpetuate beneficial immune responses in the elderly.

Interleukin-10 promotes Mycobacterium tuberculosis disease progression in CBA/J mice.

IL-10 is a potent immunomodulatory cytokine that affects innate and acquired immune responses. The immunological consequences of IL-10 production during pulmonary tuberculosis (TB) are currently unknown, although IL-10 has been implicated in reactivation TB in humans and with TB disease in mice. Using Mycobacterium tuberculosis-susceptible CBA/J mice, we show that blocking the action of IL-10 in vivo during chronic infection stabilized the pulmonary bacterial load and improved survival. Furthermore, this beneficial outcome was highly associated with the recruitment of T cells to the lungs and enhanced T cell IFN-gamma production. Our results indicate that IL-10 promotes TB disease progression. These findings have important diagnostic and/or therapeutic implications for the prevention of reactivation TB in humans.

DM, but not cathepsin L, is required to control an aerosol infection with Mycobacterium tuberculosis.

Antigen presentation by class II MHC molecules in the uninfected host is a multi-step process involving key functions provided by specific cathepsins (Cat) and the peptide editor DM. Herein, we examined the requirement for each of these components in mice to control a low-dose aerosol infection with Mycobacterium tuberculosis (MTB). Mice lacking Cat B, -L, or -S were similar to wild-type in their ability to control the growth and dissemination of MTB. In contrast, DM(-/-) mice failed to limit MTB growth and showed approximately 100-fold higher bacterial burden in the lung and spleen (5-6 weeks postinfection) as compared with wild-type and Cat-deficient mice. Histopathology revealed impaired cellular recruitment and altered granuloma formation in the lungs of MTB-infected DM(-/-) mice. Moreover, despite impaired thymic selection in Cat L(-/-) and DM(-/-) mice, MTB-specific CD4(+) T cells were elicited only in the former. The lower numbers of MTB-specific CD4(+) T cells available in Cat L(-/-) mice as compared with wild-type animals were sufficient to control MTB growth and dissemination. In addition, DM(-/-) macrophages infected with MTB in vitro were unable to stimulate pathogen-specific T cells. The data indicate that the majority of antigens derived from MTB are loaded onto nascent class II MHC molecules via the classical DM-dependent pathway.

Peripheral blood gamma interferon release assays predict lung responses and Mycobacterium tuberculosis disease outcome in mice.

Current diagnostic tests for tuberculosis (TB) are not able to distinguish active disease from latent Mycobacterium tuberculosis infection, nor are they able to quantify the risk of a latently infected person progressing to active TB. There is interest, however, in adapting antigen-specific gamma interferon (IFN-gamma) release assays (IGRAs) to predict disease outcome. In this study, we used the differential susceptibilities of inbred mouse strains to M. tuberculosis infection to evaluate the prognostic capabilities of IGRAs. Using lung and blood cultures, we determined that CBA/J, DBA/2, and C3H/HeJ mice (models of heightened risk of progression to active TB) produced less antigen-specific IFN-gamma in response to M. tuberculosis culture filtrate proteins and early secreted antigenic target-6 than the relatively resistant C57BL/6 mouse strain. Additionally, reduced IFN-gamma secretion in supernatants reflected a reduced frequency of IFN-gamma-responding cells in the lung and blood and not a specific defect in IFN-gamma secretion at the single-cell level. Importantly, detection of antigen-specific IFN-gamma from blood cultures accurately reflected lung responses, indicating that blood can be an appropriate test tissue in humans. Furthermore, reduced antigen-specific IFN-gamma production and low frequencies of IFN-gamma-responding cells from peripheral blood predicted increased risk of TB disease progression across genetically diverse TB disease-susceptible mouse strains, suggesting that similar results may occur in humans. The development of efficacious predictive diagnostic tests for humans would lead to targeted therapy prior to progression to active TB, reducing transmission, incidence, and prevalence rates while maximizing the use of public health resources.

Age dependent increase in early resistance of mice to Mycobacterium tuberculosis is associated with an increase in CD8 T cells that are capable of antigen independent IFN-gamma production.

The lungs of naïve 18-month-old mice contain an abundant resident population of CD8 T cells that express typical markers of memory, express elevated levels of Th1 cytokine receptors on their surface, and are capable of non-specific IFN-gamma production in response to a Th1 cytokine cocktail. In this study we characterize this population of CD8 T cells in the lungs and spleens of mice with increasing age. In general, the proportion of CD8 T cells expressing markers of memory and Th1 cytokine receptors increased with age. The enhanced ability of CD8 T cells to produce IFN-gamma in an antigen independent manner followed this pattern as well, beginning to increase between 6 and 12 months of age. Interestingly, the phenotypic and functional age-related changes in CD8 T cells were also associated with a progressive age-related increase in early resistance to Mycobacterium tuberculosis. Taken together, these data suggest that as mice age a population of memory CD8 T cells, that are capable of contributing to innate immune responses to M. tuberculosis, gradually emerges and could be relevant for developing strategies to enhance immunity in the elderly.

Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination.

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.

Th1 cytokines facilitate CD8-T-cell-mediated early resistance to infection with Mycobacterium tuberculosis in old mice.

Numerous immunological defects begin to emerge as an individual ages, the consequence of which is heightened susceptibility to infectious diseases. Despite this decline in immune function, old mice display an early transient resistance to Mycobacterium tuberculosis infection in the lung, which is dependent on CD8 T cells and gamma interferon (IFN-gamma) production. In this study, we investigated the mechanism of resistance by examining the CD8-T-cell phenotype and function in old naïve and M. tuberculosis-infected mice. Pulmonary CD8 T cells from naïve old mice expressed cell surface markers of memory in addition to receptors for several Th1 cytokines. Stimulation of lung cells from naïve old mice with a combination of Th1 cytokines (interleukin-2 [IL-2], IL-12, and IL-18) resulted in nonspecific production of IFN-gamma by memory CD8 T cells. Following aerosol infection with M. tuberculosis, the lungs of old mice contained significantly more IL-12, IL-18, and IFN-gamma than the lungs of young mice contained. Together, these data demonstrate that the increased and early production of Th1 cytokines in the lungs of M. tuberculosis-infected old mice, in combination with CD8 T cells that can nonspecifically produce IFN-gamma, leads to transient control of M. tuberculosis growth in the lungs of old mice. Further characterization of this mechanism should provide essential information regarding the aging immune system and should contribute to the development of novel strategies to decrease the morbidity and mortality of the aging population associated with infectious diseases.

The influence of age on immunity to infection with Mycobacterium tuberculosis.

Changes within the immune system during aging lead to an elderly population that is both highly susceptible to infectious diseases and unresponsive to typical vaccine protocols. Using the murine model of tuberculosis, we have identified key differences in the generation of T-helper 1 cell immunity between old and young mice, and this information may be important for the design of new vaccines or post exposure therapies to protect the elderly against infectious diseases. In response to infection with Mycobacterium tuberculosis, it has been shown that the generation of antigen-specific CD4(+) T-cell immunity is impaired in old mice. In contrast, recent findings document that old mice display a transient enhanced resistance that occurs within the first 3 weeks of infection. Early resistance was associated with the presence of CD8(+) T cells and their ability to produce interferon-gamma (IFN-gamma) well before their young counterparts. Further investigation into the mechanism by which CD8(+) T cells are induced to secrete IFN-gammain vivo could provide an approach to enhance the effector function of these cells and subsequently protect elderly individuals from respiratory pathogens such as M. tuberculosis.