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

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Abstract

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.

Introduction

The elderly are highly susceptible to many infectious diseases, and current vaccines are only moderately protective in this specific age group (Cabre, 2009, Grubeck-Loebenstein et al., 2009, Sambhara and McElhaney, 2009). There is a critical need to develop improved vaccines that specifically target an aged immune system. Recent evidence suggests that the inclusion of toll like receptor (TLR) agonists can serve as appropriate vaccine adjuvants that drive the production of pro-inflammatory cytokines and lead to enhanced protection (Baldridge et al., 2004, Lahiri et al., 2008). Promising studies have been performed using young animals however very little consideration has been made for whether these new vaccines will effectively target an aged immune system. Indeed, TLR expression and function in aged hosts has been examined in splenic and peritoneal macrophages from old mice, with a decrease in TLR mRNA levels and ligand induced cytokine production relative to similar cells from young mice (Renshaw et al., 2002). In addition, TLR-2/1 induced TNF production was decreased in monocytes from elderly individuals (van Duin et al., 2007), thus indicating that aged mice and humans can display diminished TLR function.

In this study we determine the capacity of pulmonary macrophages (CD11c+ cells) from aged mice to respond to infection with the virulent pathogen Mycobacterium tuberculosis (M. tb). M. tb is known to interact with macrophages predominantly via TLR-2 in young mice (Underhill et al., 1999a, Underhill et al., 1999b) and in man, with additional contributions by TLR-9 and TLR-4 (Reiling et al., 2002, Bafica et al., 2005). As such, M. tb is an ideal experimental tool to dissect macrophage–TLR interactions in old age. Furthermore, tuberculosis (TB) is especially problematic in the elderly population, with individuals over the age of 65 having the highest TB case rate compared to other age groups (WHO, 2008) and highly susceptible to TB associated death (Teale et al., 1993), making study particularly relevant to public health. Understanding the function of TLRs in the lungs of aged hosts will provide insight into the increased susceptibility of the elderly to M. tb infection, and could be critical for the effective design of vaccines that specifically target the aged population.

Our studies demonstrate that pulmonary CD11c+ cells from old mice are fully capable of producing IL-12p40 and TNF following in vitro and in vivo M. tb infection however, unlike CD11c+ macrophages from young mice, M. tb induced cytokine production occurred independently of TLR-2. Blocking studies showed that TLR-4 and TLR-9 could only partially compensate for the lack of TLR-2 responsiveness, indicating that additional receptors participate in M. tb recognition in old age. These data provide evidence that the incorporation of TLR-2 agonists into vaccines may not be an effective strategy for protecting the elderly and that other, currently uncharacterized receptors, may be alternate candidates when designing vaccines specifically for aged individuals.

Section snippets

Mice

Specific-pathogen-free, female, C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) at 2 months of age (young), or at 18 months of age (old) through a contract with the National Institute On Aging. Female wild type and TLR-2 deficient C57BL/6 retired breeders were purchased from The Jackson Laboratory (Bar Harbor, ME) at approximately 9 months of age, and aged in house to 14–18 months of age. Mice were housed in a standard vivarium in microisolator cages and were

M. tb induced cytokine production by pulmonary CD11c+ cells remains intact in old age

We determined the capacity of pulmonary macrophages from old and young mice to respond to M. tb infection by secreting IL-12p40 and TNF, which are both produced upon TLR ligation and critical for the control of M. tb. Our initial studies compared the myeloid cell populations within the lungs of old and young naïve mice which revealed that CD11b expression was similar, but the proportion of CD11c+ cells was consistently increased in the lungs of young mice (average of 5% more compared to old;

Discussion

In this study we determined the capacity of pulmonary macrophages from old and young mice to produce IL-12p40 and TNF in response to M. tb infection. Our findings demonstrate that pulmonary CD11c+ cells (a marker associated with alveolar macrophages (Gonzalez-Juarrero et al., 2003)) were the primary cells that could secrete IL-12p40 and TNF both in vitro and in vivo in response to M. tb. This response was equivalent between young and old mice, indicating that early cytokine secretion by

Acknowledgements

The project described was supported by grant number R01AG-021097 from the National Institute on Aging, and 1T32HL07946-06 from the National Institute of Health. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institute on Aging or the National Institute of Health.

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    Current address: International Laboratory Branch, Division of Global AIDS, Centers for Disease Control and Prevention, 1600 Clifton Rd NE Mailstop A-11, Atlanta, GA 30333, United States.

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